TESE_Efeitos da terapia probiótica (Bacillus subtilis)

RENATA DE CARVALHO FOUREAUX

EFEITOS DA TERAPIA PROBIÓTICA ( Bacillus

subtilis) SOBRE PARÂMETROS METABÓLICOS

E INFLAMATÓRIOS EM RATOS COM

PERIODONTITE INDUZIDA POR LIGADURA

ASSOCIADA OU NÃO AO ESTRESSE CRÔNICO

LAVRAS – MG

2014

RENATA DE CARVALHO FOUREAUX

EFEITOS DA TERAPIA PROBIÓTICA ( Bacillus subtilis) SOBRE

PARÂMETROS METABÓLICOS E INFLAMATÓRIOS EM RATOS

COM PERIODONTITE INDUZIDA POR LIGADURA ASSOCIADA OU

NÃO AO ESTRESSE CRÔNICO

Tese apresentada à Universidade Federal de Lavras, como parte das exigências do Programa de Pós-Graduação em Ciências Veterinárias, área de concentração em Ciências Veterinárias, para obtenção do título de Doutor.

Orientador

Dr. Luciano José Pereira

Coorientadores

Dr. Márcio Gilberto Zangerônimo

Dr. Raimundo Vicente de Sousa

LAVRAS – MG

2014

Foureaux, Renata de Carvalho. Efeitos da terapia probiótica (Bacillus subtilis) sobre parâmetros metabólicos e inflamatórios em ratos com periodontite induzida por ligadura associada ou não ao estresse crônico / Renata de Carvalho Foureaux. – Lavras : UFLA, 2014.

108 p. : il. Tese (doutorado) – Universidade Federal de Lavras, 2014. Orientador: Luciano José Pereira. Bibliografia. 1. Modelos animais. 2. Estresse. 3. Periodontite. 4. Ligadura. 5.

Perda de inserção periodontal. I. Universidade Federal de Lavras. II. Título.

CDD – 636.0897632519.53

Ficha Catalográfica Elaborada pela Coordenadoria de Produtos e Serviços da Biblioteca Universitária da UFLA

RENATA DE CARVALHO FOUREAUX

EFEITOS DA TERAPIA PROBIÓTICA ( Bacillus subtilis) SOBRE

PARÂMETROS METABÓLICOS E INFLAMATÓRIOS EM RATOS

COM PERIODONTITE INDUZIDA POR LIGADURA ASSOCIADA OU

NÃO AO ESTRESSE CRÔNICO

Tese apresentada à Universidade Federal de Lavras, como parte das exigências do Programa de Pós-Graduação em Ciências Veterinárias, área de concentração em Ciências Veterinárias, para obtenção do título de Doutor.

APROVADA em 27 de outubro de 2014. Dr. Leandro Silva Marques UFVJM Dr. Luciano José Pereira UFLA Dr. Michel Reis Messora USP Dr. Raimundo Vicente de Sousa UFLA Dr. Saul Martins de Paiva UFMG

Dr. Luciano José Pereira

Orientador

LAVRAS – MG

2014

AGRADECIMENTOS

A Deus, por me iluminar e amparar em todos os momentos da minha

existência.

A Nossa Senhora Aparecida que me cobriu com seu Manto Sagrado nos

momentos de aflição.

À minha Mãe Guacira e meu Pai João Luiz (in memorian), pelo amor

incondicional.

À minha Irmã Guacira pelo carinho em todos os momentos.

A toda família Carvalho e toda a família Foureaux pelo incentivo.

Ao meu querido marido Lamartini, por todo seu carinho e dedicação

desde o primeiro dia da nossa vivência.

Ao querido orientador “Pai” Prof. Luciano José Pereira pela infinita

dedicação na busca do aprimoramento dos meus conhecimentos. Agradeço por

toda vida seus conselhos, amizade, carinho e horas dedicadas a nossa pesquisa.

Obrigada também Julieta, Alice e Lucas por compreenderem a ausência nos

momentos dos nossos estudos.

Ao Prof. Raimundo Vicente de Sousa, que foi meu primeiro

incentivador dessa empreitada. Sem suas palavras de estímulo nunca poderia ter

chegado até aqui.

Ao Prof. Michel R. Messora e ao Prof. Marcelo Henrique Napimoga por

todo auxílio e incentivo.

Aos membros da banca Prof. Leandro Silva Marques e Prof. Saul

Martins de Paiva por sua disponibilidade no aprimoramento deste trabalho.

À Universidade Federal de Lavras, na pessoa de seu Reitor José Roberto

Soares Scolforo, por me proporcionar a obtenção da luz do conhecimento.

Ao Departamento de Ciências Veterinárias, seus Professores e ao

Coordenador do Programa de Pós-Graduação Dr. Márcio Gilberto Zangerônimo.

Aos funcionários do Departamento de Medicina Veterinária, Willian

César Cortez e José Reinaldo dos Reis Ferreira “Berin”, pelo carinho e

dedicação.

À reitora do UNILAVRAS, professora Christiane Amaral Lunkes

Argenta e ao professor Cássio Vicente Pereira pelo auxílio na condução deste

projeto.

Aos colegas Eric Francelino Andrade, Ticiana Vasques, Matheus Soares

da Silva Ferreira, Viviam de Oliveira Silva, Luiz Fernando Ferreira de Oliveira,

Thiago Santos Araújo, Thiago da Silva Teófilo, Priscila Faria Rosa Lopes, Edna

Lopes, César Augusto Pospissil Garbossa pela paciência e companheirismo.

Aos colegas cirurgiões-dentistas Dr. Paulo Murilo Laurente, Dr. Mário

César Mazzini, Dr. Antônio Adolfo Gattini Corsini Sbampato e Dr. Carlos

Rogério O. Leite pelo apoio.

Às amigas Luciana Botezelli e Geysiane Mara Custódio Mesquita pelas

palavras nos momentos de angústia e também nos de alegria.

A minha querida secretária Endrika Thomaz por seu carinho e

dedicação.

Às minhas cachorrinhas Flor e Linda e ao Juan, pelas horas de

companhia e carinho em todo este tempo.

Agradecimentos especiais ao Conselho Nacional de Pesquisa e

Desenvolvimento Tecnológico e à Fundação de Amparo à Pesquisa do Estado de

Minas Gerais (FAPEMIG) pelo auxílio financeiro concedido (CNPq - Processo

475468/2010-4e FAPEMIG – Processo CDS - APQ-01692-12).

“Sonhe com aquilo que você quiser. Seja

o que você quer ser, porque você possui

apenas uma vida e nela só se tem uma chance

de fazer aquilo que se quer. Tenha felicidade

bastante para fazê-la forte. Tristeza para fazê-la

humana. E esperança para fazê-la feliz. A

felicidade aparece para aqueles que choram.

Para aqueles que se machucam. Para aqueles

que buscam e tentam sempre. E para aqueles

que reconhecem a importância das pessoas

que passam por suas vidas.”

Clarice Lispector

RESUMO

A doença periodontal é caracterizada por um processo inflamatório crônico dos tecidos de suporte dos dentes, dependente da resposta do hospedeiro, em reação à presença de periodontopatógenos no biofilme subgengival. Medidas locais e sistêmicas de modulação da resposta imunológica e do ambiente microbiano têm sido investigadas. Nesse contexto surgem os probióticos que são organismos vivos que quando administrados em quantidades adequadas conferem benefícios à saúde, despertando o interesse de sua aplicação na terapia periodontal. O presente estudo foi dividido em dois capítulos. No primeiro, foi feita uma revisão sistemática avaliando-se a eficácia dos probióticos na resposta imune em ratos submetidos a desafios experimentais. Os trabalhos foram pesquisados em três bases Pubmed, ISI Web of Science e Scielo. As espécies bacterianas mais empregadas foram os Lactobacillus e Bifidobacterium. Oitenta e seis porcento dos artigos selecionados mostraram efeito benéfico na resposta imune associado ao uso de probióticos. No segundo capítulo foi avaliado o efeito da terapia probiótica em ratos com indução de doença periodontal associada ao estresse de restrição. O delineamento experimental foi inteiramente casualizado em esquema fatorial 2x2x2 (com e sem doença periodontal (DP), com e sem estresse crônico (EC), com e sem o uso de probióticos) com seis repetições em cada grupo. O probiótico Bacillus subtilis foi administrado oralmente na proporção de 1,5x108 unidades formadoras de colônia (UFC)/mL na água de bebida dos animais durante 45 dias. O EC foi realizado por meio de imobilização diária por 2,5 horas a partir do 16º dia durante 30 dias, enquanto que a DP foi induzida com o protocolo de ligadura nos primeiros molares mandibulares direito e esquerdo a partir do 31º dia por 14 dias. Foram medidas a altura de vilosidades e profundidade de criptas do duodeno, jejuno e íleo. A eutanásia foi feita no 45º dia do experimento. O nível de significância foi fixado em p <0,05. A DP aumentou a perda óssea alveolar, os níveis de ciclooxigenase-2 (COX-2), telopeptídeo carboxiterminal do colágeno tipo-1 (CTX), p38 quinases ativadas por mitógeno (MAPK), ligante do receptor ativador da NF-κB (RANKL) e diminuiu os níveis de osteoprotegerina (OPG) (p <0,05). Os ratos estressados apresentaram níveis mais altos de peptídeo-C, corticosterona e glicose (p <0.05). A presença do estresse reduziu a expressão de CTX e p38 (p<0.05). O probiótico reduziu a perda óssea alveolar nos ratos não estressados, bem como diminuiu a expressão do CTX e induziu ao aumento da expressão da OPG em ratos não estressados com DP (p <0,05). Embora o probiótico não tenha sido efetivo na prevenção da perda óssea ou alteração da expressão dos marcadores inflamatórios em ratos estressados, o número de células inflamatórias diminuiu (p <0.05). Os grupos com estresse e

DP apresentaram diminuição na altura das vilosidades e profundidade de criptas intestinais (p <0,05). Em conclusão geral, os probióticos foram benéficos na modulação da resposta imune e se mostraram promissores no controle da doença periodontal. Palavras-chave: Modelos animais. Estresse. Periodontite. Ligadura. Perda de inserção periodontal.

ABSTRACT

Periodontal disease is characterized by chronic inflammation of the tissues supporting the teeth, depending on the host response, in reaction to the presence of periodontal pathogens in sub-gingival biofilm. Local and systemic measures modulation of the immune response and of microbial environment has been investigated. In this context emerge probiotics which are live organisms that when administered in adequate amounts give beneficial health effects, arousing the interest of its application in periodontal therapy. The present study was divided in two chapters. In the first, it was made a systematic review evaluating the probiotics effect on immune response in rats submitted to different experimental challenges. The survey was using PubMed, ISI Web of Science and Scielo databases. The bacteria species most used were Lactobacillus and Bifidobacterium. Eighty-six percent of the selected articles reported a beneficial effect on the immune response associated to the probiotics use. In the second chapter it was evaluated the probiotic therapy effect in rats with periodontal disease induction associated with restriction stress. The experimental design was completely randomized factorial 2x2x2 (with and without periodontal disease (PD), with and without chronic stress (CS), with and without probiotics use) with six replicates in each group. The probiotic Bacillus subtilis was administered orally at 1.5 x 108 proportion of colony-forming units (CFU)/mL to the drinking water of the animals for 45 days. The CS was performed by immobilization daily for 2.5 hours from the 16th day during 30 days, while the PD was induced by the ligation protocol in the first right and left mandibular molars from 31st day for 14 days. Villus height and crypt depth of the duodenum, jejunum and ileum were measured. Euthanasia was performed on the 45th day of the experiment. The significance level was set at p<0.05. PD increased alveolar bone loss, levels of cyclooxygenase-2 (COX-2), carboxyterminal telopeptide of collagen type-1 (CTX), p38 mitogen-activated kinases (MAPK), ligand receptor activator of NF-κB (RANKL) and decreased levels of osteoprotegerin (OPG) (p<0.05). The stressed rats had higher levels of C-peptide, corticosterone and glucose (p<0.05). The presence of stress decreased expression of p38 and CTX (p<0.05). The probiotic reduced alveolar bone loss in the non-stressed rats, as well as decreased expression of CTX and induced the increased expression of OPG in unstressed rats with PD (p<0.05). Although the probiotic has not been effective in preventing bone loss or alteration of the expression of inflammatory markers in stressed rats, the number of inflammatory cells decreased (p<0.05). Groups with stress and PD showed decrease in villous height and depth of intestinal crypts (p<0.05). In general conclusion, probiotics were beneficial in the modulation of immune response and have a promising in the control of periodontal disease. Keywords: Animal models. Stress. Periodontitis. Ligature. Periodontal attachment loss.

LISTA DE ILUSTRAÇÕES

SEGUNDA PARTE - ARTIGOS

ARTIGO 2

Figure 1 Time schedule of experimental period........................................... 97 Figure 2 Means and standard deviations of plasmatic corticosterone, C-

peptide and glucose levels............................................................. 98 Figure 3 Means and standard deviations of CTX, Cox-2, p-38, RANK-L,

OPG expression and number of inflammatory cells evaluated on periodontal tissues ........................................................................ 99

LISTA DE TABELAS

SEGUNDA PARTE - ARTIGOS

ARTIGO 1

Table 1 Summary of the Selected Studies……………………………………67 Table 2 Evaluation criteria and scores for the selected articles……...………70

ARTIGO 2

Table 1 Mean values of morphometric analysis rat mandible ......................100 Table 2 Means and standard deviations of the villous height and crypt depth

in small intestine sections, with comparisons among groups...........101

LISTA DE ABREVIATURAS E SIGLAS

ACTH Hormônio Adrenocorticotrófico

DP Doença periodontal

CH201 Bioplus 2B - CH Hansen (Hørsholm, Denmark)

cm Centímetro

CO Crista Óssea

COX-1 Ciclooxigenase-1

COX-2 Ciclooxigenase-2

CRF Hormônio de Liberação de Corticotropina

CTX Telopeptídeo Carboxiterminal do Colágeno Tipo 1

DCR Doença Crônica Renal

EC Estresse crônico

EDTA Ácido etilenodiamino tetra-acético

FAO Organização das Nações Unidas para Alimentação e

Agricultura

HPA Eixo hipotálamo-hipófise-adrenal

IBD Doença Inflamatória do Intestino

IFN-γ Interferon-gama

Ig Imunoglobulina

IL Interleucina

JEC Junção Cemento-Esmalte

LPS Lipopolisacarídeos

kg Quilograma

kV Quilovolts

mA Miliampere

MAPK Quinases ativadas por mitógeno

mg Miligrama

MMP Matriz Metaloproteinase

NFκβ Fator Nuclear kappa Beta

NK Células Natural Killer

OPG Osteoprotegerina

PGE2 Prostaglandina E2

pH Potencial Hidrogeniônico

PIE Perda de Inserção Epitelial

PMN Polimorfonucleares

POA Perda Óssea Alveolar

RAR Raspagem e Alisamento Radicular

RNA Ácido Ribonucleico

RANK Receptor ativador do fator kappa B (NF-κβ)

RANKL Ligante do Receptor ativador do fator kappa B (NF-κβ)

SOP Suporte Ósseo Periodontal

SP Substância P

TIMPs Inibidores de Metaloproteinases Teciduais

TNF-α Fator de Necrose Tumoral alfa

UFC Unidades Formadoras de Colônias

VIP Peptídeo Vasoativo Intestinal

WHO Organização Mundial de Saúde

SUMÁRIO

PRIMEIRA PARTE ...........................................................................17 1 INTRODUÇÃO ..................................................................................17

2 REFERENCIAL TEÓRICO ..............................................................20 2.1 Doença periodontal.............................................................................20 2.2 Modelos experimentais de doença periodontal..................................24

2.3 Estresse e Doença periodontal............................................................28 2.4 Probióticos e doença periodontal.......................................................31 2.5 Probióticos e imunidade.....................................................................34 3 CONSIDERAÇÕES GERAIS............................................................35 REFERÊNCIAS .................................................................................36

SEGUNDA PARTE – ARTIGOS.......................................................46 ARTIGO 1 Effect of Probiotic Administration on the Immune

Response: A Systematic Review of Experimental Models in Rats.....46 1 INTRODUCTION ..............................................................................48 2 MATERIALS AND METHODS ........................................................50 3 RESULTS...........................................................................................53

4 DISCUSSION.....................................................................................56

5 CONCLUSION...................................................................................60

REFERENCES...................................................................................61

ARTIGO 2 Effects of probiotic therapy on metabolic and inflammatory parameters of rats with ligature-induced periodontitis associated with restraint stress.....................................73

1 INTRODUCTION ..............................................................................77 2 MATERIALS AND METHODS ........................................................79 3 RESULTS...........................................................................................84

4 DISCUSSION.....................................................................................86

5 CONCLUSIONS.................................................................................90

REFERENCES...................................................................................91

ANEXOS ........................................................................................... 102

17

PRIMEIRA PARTE

1 INTRODUÇÃO

A doença periodontal é caracterizada por um processo inflamatório

crônico dos tecidos de suporte dos dentes, dependente da resposta do

hospedeiro, em reação à presença de periodontopatógenos no biofilme

subgengival (HAYTAC et al., 2014). A etiologia da forma mais comum de DP –

a gengivite - está relacionada à presença de biofilme microbiano (estrutura bem

organizada de bactérias) aderido a superfície dentária (TERHEYDEN et al.,

2014). As principais espécies patógenas que compõem o biofilme dentário e que

determinam as várias formas de periodontite são: Porphyromonas gingivalis,

Prevotella intermedia, Actinobacillus actinomycetemcomitans, Fusobacterium

nucleatum, entre outros (SOCRANSKY et al., 1998).

Adicionalmente aos microrganismos existentes na cavidade bucal, o

estresse pode ter efeitos negativos sobre o sistema imunológico e/ou mediar

efeitos comportamentais nas defesas do organismo, contribuindo sobremaneira

na etiologia e perpetuação da periodontite crônica (BOYAPATI; WANG, 2007).

O estresse pode alterar a resistência tecidual do hospedeiro por mecanismos

autonômicos e endócrinos, resultando principalmente na elevação dos níveis de

corticosteroides e catecolaminas, reduzindo a microcirculação da gengiva e do

fluxo salivar, com relatos de redução das funções de neutrófilos e linfócitos, o

que facilita a invasão bacteriana e o dano tecidual (BOYAPATI; WANG, 2007;

HORNING; COHEN, 1995; JOHNSON; ENGEL, 1986).

A remoção do biofilme e diminuição do número de agentes bacterianos

por meios físicos, como: escovação, raspagem e alisamento coronoradicular,

irradiação laser, fototerapia (ALMEIDA et al., 2007; CARVALHO et al., 2011;

LOPES et al., 2010) e químicos locais - clorexidina, óleos essenciais

18

(HAFFAJEE et al., 2009; KRAYER et al., 2010) são utilizados no tratamento

convencional da periodontite crônica. Porém, outras medidas, tais como

antibioticoterapia local ou sistêmica também têm sido empregadas em casos

graves especialmente (FERNANDES et al., 2010). Mas, o desenvolvimento da

resistência aos antibióticos por certos patógenos tem aumentado a possibilidade

do retorno a uma época pré-antibióticos (HAYTAC et al., 2014).

Portanto, uma vez que o processo inflamatório é dependente da reação

imunológica individual frente à presença do biofilme, uma resposta inadequada

ou excessiva a agressão bacteriana é também fator preponderante na progressão

da doença periodontal (DOMON et al., 2014; TAKAHASHI et al., 2014;

TERHEYDEN et al., 2014). O controle da resposta imunológica e do ambiente

microbiano, como por exemplo, o emprego de probióticos, tem surgido como

terapia adjuvante no controle da doença periodontal (DEVINE; MARSH, 2009).

Esses podem promover uma defesa natural contra bactérias patógenas, o que

pode ser favorável para manutenção da saúde bucal. Porém, a identificação de

probióticos úteis, o estabelecimento da dose mais apropriada e o veículo de seu

uso necessitam de mais investigações (HAYTAC et al., 2014).

Nos sistemas imune e inflamatório a comunicação celular é organizada

por proteínas, peptídeos ou moléculas mensageiras chamadas citocinas. Essas

moléculas interagem seletivamente com receptores transmembranas, ativando

outros mensageiros intracelulares que amplificam, transportam o sinal ao núcleo

celular, onde modulam a atividade gênica e síntese proteica (TERHEYDEN et

al., 2014). O entendimento dessas vias de sinalização na imunologia da

periodontite é fundamental para o entendimento das citocinas e quimiocinas

envolvidas na comunicação celular e na defesa do organismo (EBERSOLE et

al., 2013) de forma a propociar e desenvolver modalidades terapêuticas

alternativas.

19

Diante do exposto, no presente estudo objetivou-se avaliar os efeitos da

ingestão de probióticos em uma revisão sistemática e seu uso em ratos Wistar

com ou sem indução da doença periodontal por ligadura associado ou não ao

estresse crônico de imobilização.

20

2 REFERENCIAL TEÓRICO

2.1 Doença periodontal

A doença periodontal afeta extensivamente as populações e é o principal

fator de risco para a perda dentária (DYE 2012; GHIZONI et al., 2012;

HUMPHREY et al., 2008; KURITA-OCHIAI et al., 2014; MACRI et al., 2014;

OLIVER et al., 1998).

Segundo a American Academy of Periodontology (2000), a gengivite

induzida pelo biofilme é definida como uma inflamação da gengiva sem a perda

da inserção epitelial. Pode ser caracterizada pela presença de alguns dos

seguintes sinais: vermelhidão e edema dos tecidos gengivais, sangramento

provocado, mudanças do contorno e consistência, presença de cálculo e/ou

biofilme e sem evidência radiográfica de perda da crista óssea. É caracterizada

pela presença de bactérias Gram-negativa, bastonetes móveis e filamentos.

A periodontite crônica com leve a moderada perda de suporte

periodontal é definida como a inflamação da gengiva extendendo aos tecidos de

suporte adjacentes. A doença é caracterizada pela perda de inserção associada à

destruição do ligamento periodontal e perda do osso de suporte adjacente. A

destruição leve à moderada é geralmente caracterizada pela sondagem das bolsas

periodontais até 6 mm com perda de inserção clínica até 4mm. As evidências

radiográficas mostram perda óssea e aumento da mobilidade dentária que pode

estar presente. Apresenta uma população predominante de bactérias Gram-

negativa, com uma grande proporção de espiroquetas (AMERICAN

ACADEMY OF PERIODONTOLOGY, 2000).

A periodontite crônica com perda avançada de suporte periodontal é

definida como inflamação da gengiva e tecidos adjacentes. A doença é

caracterizada pela perda de inserção clínica acompanhada pela destruição do

21

ligamento periodontal e perda do tecido ósseo adjacente de suporte. A destruição

é geralmente caracterizada pela sondagem das bolsas periodontais maiores que

6mm com perda de inserção maior que 4mm. A evidência radiográfica de perda

óssea é aparente e o aumento da mobilidade pode estar presente. Apresenta uma

população predominante de bactérias Gram-negativa, com uma grande

proporção de espiroquetas (AMERICAN ACADEMY OF

PERIODONTOLOGY, 2000). Na periodontite crônica A.

actinomycetemcomitans e P. gingivalis mostraram ser capazes de invadir o

epitélio gengival in vitro (FERES et al., 2004).

A periodontite agressiva envolve diferentes tipos de periodontia que

afetam as pessoas que, na maioria dos casos, podem parecer saudáveis. Pode ser

familiar e há uma rápida taxa de progressão da doença. A periodontite agressiva

ocorre de forma localizada e generalizada (AMERICAN ACADEMY OF

PERIODONTOLOGY, 2000).

A periodontite agressiva localizada, usualmente, tem um começo

circumpubertal com o início da destruição periodontal localizado nos primeiros

molares permanentes e incisivos, embora padrões atípicos de dentes afetados

serem possíveis. A doença é frequentemente associada com o patógeno

periodontal Actinobacillus actinomycetemcomitans e anomalias das funções dos

neutrófilos, além de coccos e bastonetes facultativos. Uma forte resposta dos

anticorpos séricos aos agentes infectantes é detectada frequentemente

(AMERICAN ACADEMY OF PERIODONTOLOGY, 2000).

A periodontite agressiva generalizada, usualmente, afeta pessoas com 30

anos ou menos, mas podem ser em mais velhos. Há pelo menos três dentes

permanentes, sem ser os primeiros molares e incisivos, afetados pela perda de

inserção interproximal. A perda de inserção ocorre em períodos pronunciados de

destruição. A doença é frequentemente associada aos patógenos periodontais

Actinobacillus actinomycetemcomitans e Porphyromonas gingivalis e anomalias

22

das funções dos neutrófilos, além de coccos e bastonetes facultativos. Uma

pobre resposta dos anticorpos séricos aos agentes infectantes é detectada

frequentemente (AMERICAN ACADEMY OF PERIODONTOLOGY, 2000).

A doença periodontal é uma infecção crônica, assintomática, com

incidência mundial e especialmente prevalente em populações mais idosas.

Relatos recentes mostram as associações e o potencial dos mecanismos

biológicos entre a doença periodontal e outras doenças sistêmicas, associando a

doença periodontal como um fator de risco para enfermidades cardíacas,

aterosclerose, doença crônica pulmonar obstrutiva, diabetes mellitus e câncer,

além de causar um estado crônico de inflamação sistêmica (BOYLAN et al.,

2014; GHIZONI et al., 2012; KURITA-OCHIAI et al., 2014; MACRI et al.,

2014; SCARABELOT et al., 2014; TSAI et al., 2014).

As bactérias Porphyromonas gingivalis e Aggregatibacter

actinomycetemcomitans, agentes periodontopatógenos muito prevalentes podem

acelerar a deposição de ateromas em modelos animais (GHIZONI et al., 2012;

KURITA-OCHIAI et al., 2014). Além dessas bactérias ainda existem outras

espécies que estão fortemente ligadas à progressão da doença periodontal, tais

como Bacteroides forsythus, Prevotella intermedia, Peptostreptococcus micros e

Fusobacterium nucleatum (ALJEHANI, 2014).

A doença periodontal é caracterizada pela destruição irreversível das

estruturas de suporte dos dentes, incluindo ligamento periodontal, osso alveolar

e tecidos gengivais, levando a um colapso do cemento pela condição

inflamatória dos tecidos moles ao redor dos dentes (KURITA-OCHIAI et al.,

2014; MACRI et al., 2014). A reabsorção óssea alveolar patológica na

periodontite é um resultado da infecção crônica com remissões e progressões,

apresentando patogênese semelhante a outras doenças que afetam os ossos

(GURSOY et al., 2013). Assim, o plano de tratamento deve incluir tanto

considerações locais como sistêmicas (TSAI et al., 2014).

23

Existem microrganismos colonizadores da pele, boca, tratos digestivo e

reprodutivo, que constituem estruturas tridimensionais de agregados de bactérias

que se unem umas as outras e também à superfície formando biofilmes.

Principalmente àqueles associados a bactérias anaeróbias gram-negativas, têm

uma participação bem estabelecida nas doenças orais, tais como nas cáries

dentária, gengivite e periodontite, existindo mais de quinhentas espécies

bacterianas distintas (KURITA-OCHIAI et al., 2014; MANCL; KIRSNER;

AJDIC, 2013). Porém, somente um pequeno número está associado à doença e

considerado etiologicamente importante (ALJEHANI, 2014). Estudos sugerem

que os agentes etiológicos específicos da doença periodontal incluem

espiroquetas, fusiformes e estreptococos (FERES et al., 2004).

Os patógenos periodontais presentes nos biofilmes microbianos iniciam

a doença difundindo produtos deletérios e enzimas, tais como hialuronidases,

colagenases e proteases que quebram as matrizes extracelulares, tais como

colágeno e membranas celulares, com o intuito de produzir nutrientes para seu

crescimento e subsequente invasão tecidual (GULATI et al., 2014), levando os

tecidos periodontais a atuar como reservatórios de endotoxinas, citocinas,

lipídeos e mediadores inflamatórios que podem afetar outras partes do

organismo (SCARABELOT et al., 2014).

A invasão bacteriana resulta em uma resposta inflamatória nos tecidos

periodontais caracterizada por uma produção de citocinas, tais como

interleucinas (IL), fator de necrose tumoral (TNF-α), prostanoides, tais como

prostaglandina E2 e enzimas incluindo as matrizes metaloproteinases (MMPs)

(ALJEHANI 2014; GULATI et al., 2014; SCARABELOT et al., 2014) e

diminuição dos níveis de osteoprotegerina (OPG) não só nos tecidos gengivais e

saliva, mas também no plasma em indivíduos afetados pela doença periodontal

(JIANG et al., 2013). Em indivíduos saudáveis, os níveis desses mediadores nos

tecidos periodontais são equilibrados pelas citocinas e enzimas anti-

24

inflamatórias do sistema imune que tem a função de eliminar os patógenos

microbianos e proteger o hospedeiro (GULATI et al., 2014).

2.2 Modelos experimentais de doença periodontal

Modelos animais podem ser utilizados para se avaliar a progressão e o

efeito de diferentes tratamentos em doença periodontal induzida (MOLON et al.,

2014; WEINBERG; BRAL, 1999), bem como os efeitos do estresse sobre a

progressão da perda óssea alveolar (TAKADA et al., 2004; ZHAO et al., 2012).

O modelo experimental de indução da doença periodontal em ratos por

ligadura com fio de algodão mostra ser adequado na obtenção das alterações na

estrutura óssea periodontal, atuando como um local de colonização bacteriana

(MOLON et al., 2014; OZ; PULEO, 2011; YAGAN et al., 2014). Os ratos

também têm várias similaridades com os humanos com relação à anatomia

periodontal, formação e composição do biofilme dental, histopatologia das

lesões periodontais e imunologia básica (CHANG et al., 2014; EBERSOLE et

al., 2013; MACRI et al., 2014), facilitando dessa forma a extrapolação dos

resultados (GENCO et al., 1999; KLAUSEN; EVANS; SFINTESCU, 1989;

MOLON et al., 2014).

O modelo experimental de indução da doença periodontal por ligadura é

caracterizado pelo acúmulo de biofilme, aplainamento e deslocamento da crista

gengival, aumento da proliferação do epitélio abaixo da linha do tecido

conjuntivo e infiltração de células mononucleares inflamatórias (ALMEIDA et

al., 2007). A inflamação associada à indução da doença periodontal por ligadura

iniciada na margem gengival é causada tanto pelo trauma mecânico bem como o

aumento do acúmulo de microrganismos no biofilme (HOLZHAUSEN et al.,

2002). O modelo experimental de indução da doença periodontal por injeção de

lipopolissacarídeo (LPS) é um método relativamente simples em que uma única

25

bactéria infecta o animal levando a destruição periodontal (NAKAJIMA et al.,

2006). O lipopolissacarídeo compreende a superfície exterior de todas as

bactérias gram-negativas subgengivais, capaz de penetrar nos tecidos

conjuntivos (CHANG et al., 2014) e pode induzir a inflamação no tecido

periodontal, podendo afetar os leucócitos circulantes diretamente e ativar os

osteoclastos (DO et al., 2013). Existem diferenças nos métodos de indução de

doença periodontal em modelo de rato na ativação da cinética das vias de

sinalização inflamatórias, dependendo do tipo de resposta do hospedeiro. A

indução por lipopolissacarídeo (LPS) foi associada com uma ativação mais lenta

das vias de sinalização comparada com o modelo de ligadura, exceto para ERK

MAPK (AQUINO et al., 2009).

A partir da indução, vários métodos podem ser empregados para

avaliação da evolução da doença periodontal, tais como os métodos radiográfico

(KESAVALU et al., 2006; REED; POLSON, 1984), morfométrico (BAKER et

al., 1983; YU et al., 2007) e histométrico (ALMEIDA et al., 2007; WOLFSON;

SELTZER, 1975).

Os sinalizadores inflamatórios podem destruir a integridade tecidual

levando ao acesso bacteriano mais profundo no tecido, podendo estar

relacionado à gravidade da doença periodontal (TSAI et al., 2014). Além disso, a

sinalização e a regulação da expressão desses marcadores inflamatórios

representam um papel crítico na remodelação óssea do periodonto (KANZAKI

et al., 2002).

No processo inflamatório ocorre a liberação de citocinas que interagem

com os tecidos (células endoteliais, macrófagos, plaquetas, entre outras),

promovendo a ativação de vias inflamatórias diversas, como por exemplo, a do

ácido araquidônico. A ativação da fosfolipase A2 pelas citocinas promove a

conversão dos fosfolipídeos da membrana em ácido araquidônico, que por sua

vez é utilizado como substrato das cicloxigenases (COX) para formação de

26

prostaglandinas e prostaciclinas que são importantes mediadores da inflamação

(MESA et al., 2012; TSAI et al., 2014). Existem duas isoformas de

cicloxigenase: COX-1 e COX-2. A COX-1 é uma enzima geralmente

constitutiva responsável pela formação de prostaglandinas nas funções

fisiológicas (proteção gastrintestinal, por exemplo), enquanto COX-2 é uma

enzima induzida principalmente por citocinas proinflamatórias envolvidas nos

processos patofisiológicos, tais como vasodilatação, aumento da permeabilidade

vascular, diapedese e migração leucocitária. As prostaglandinas são potentes

estimuladores da formação e reabsorção óssea e são produzidas pelos

osteoblastos e células do ligamento periodontal (KAYAL et al., 2013). A

prostaglandina E2 (PGE2) é associada como potente estimuladora na reabsorção

óssea e perda de inserção epitelial na doença periodontal (TSAI et al., 2014). A

expressão de COX-2 nos tecidos gengivais tem sido relacionada à severidade da

doença periodontal e inflamação gengival, indicando um papel importante dessa

proteína na patogenia da periodontite crônica (MESA et al., 2012).

O receptor ativador do fator kappa B (RANK), o ligante do receptor

ativador do fator kappa B (RANKL) e osteoprotegerina (OPG) são citocinas

pertencentes à super família de fator de necrose tumoral alfa (TNFα) (KAYAL

et al., 2013). O RANK está presente nos precursores de osteoclastos e é capaz de

iniciar a sinalização da transdução osteoclastogênica, depois da ligação com

RANKL ou com o agonista anti-RANK. O RANK é um receptor capaz de

mediar a função do RANKL durante a homeostasia do osso normal e na doença

e nenhum outro receptor para RANKL foi identificado.

O RANKL é produzido pelas células T ativadas e sua expressão é

regulada para cima por muitos fatores solúveis que afetam a reabsorção óssea,

incluindo citocinas pró-inflamatórias. O RANKL é um fator crítico no sistema

imune como um importante coestimulador na ativação das células T, aparecendo

também como um elo entre a inflamação e a perda óssea (EBERSOLE et al.,

27

2013; KANZAKI et al., 2002). Esse sistema é regulado pela osteoprotegerina

(OPG) que neutraliza a habilidade do RANKL se ligar com RANK e induzir o

sinal para diferenciação dos osteoclastos (DOMON et al., 2014). A via

OPG/RANKL é a chave reguladora do metabolismo ósseo e seu efeito produz o

desenvolvimento e ativação ou não dos osteoclastos.

Ainda no processo inflamatório periodontal, o telopeptídeo do colágeno

tipo I de ligação cruzada carboxiterminal (CTX) é um marcador bioquímico de

reabsorção e formação óssea (FLEISHER et al., 2010; KWON et al., 2009).

Trata-se de um produto da degradação do colágeno usado como medidor de

reabsorção óssea (FLEISHER et al., 2010).

Outros fatores de transcrição, tais como fator nuclear �B (NF- �B),

proteína ativadora-1 (AP-1) e p38 são expressos em quantidade aumentada no

processo inflamatório, estimulando a produção de várias citocinas, muitas delas

direta ou indiretamente estimuladoras da formação dos osteoclastos (KAYAL,

2013).

As quinases ativadas por mitógeno (MAPKs) são uma família evolutiva

e mediam processos fundamentais biológicos e respostas celulares a diferentes

estímulos extracelulares por receptores múltiplos. As três principais subfamílias

de MAPKs são quinases reguladoras extracelulares (ERK-1/-2), quinases

ativadoras do c-Jun N-terminal (JNK) e p38. Os lipopolissacarídeos (LPS)

facilitam a ativação dessas três principais subfamílias (FUJITA et al., 2014). A

ativação da p38 leva ao aumento da expressão de vários genes de citocinas pela

modulação de ambos os mecanismos transcricional e pós- transcricional. A

contribuição de cada mecanismo na mudança global da expressão gênica varia

28

de acordo com o tipo de células e natureza do estímulo externo, mas os genes

que são modulados pelos mecanismos pós-transcricionais envolvendo a

modificação das proteínas que se ligam ao RNA como substratos para p38 são

TNF-α, IL-8, IL-6, IL-2 e ciclooxigenase-2 (FUJITA et al., 2014; LIANG et al.,

2014; SOUZA et al., 2012; TRAVAN et al., 2013).

A sinalização da MAPK p38 é requerida na inflamação e associada à

perda óssea decorrente de doença periodontal nos modelos animais, com uma

significativa correlação positiva na severidade da doença. Os sinalizadores

MAPK são vitais para a síntese e amplificação de mediadores pró-inflamatórios

e matriz metaloproteinases pelas células sinoviais, quimioatração de células

mononucleares e angiogênese de células endoteliais, bem como a apoptose de

células sinoviais. Assim, a p38 parece estar predominantemente envolvida nos

processos inflamatórios (TRAVAN et al., 2013).

2.3 Estresse e doença periodontal

O estresse é um estado de tensão fisiológico ou psicológico causado por

estímulos adversos, tais como físico, mental ou emocional, interno ou externo,

que tende a perturbar o funcionamento do organismo (GOYAL et al., 2013). O

fator socioeconômico, tipo de ocupação, rotina, competitividade no trabalho,

distúrbios emocionais podem levar ao aumento dos níveis de estresse. Em

modelos animais existe a influência de alguns fatores como imobilização,

temperaturas adversas, restrição, conflitos na ordem social, carência de água ou

comida, manipulação, acomodações na gaiola (umidade e inclinação), entre

outros e têm um efeito direto no eixo hipotálamo-hipófise-adrenal (SUTANTO;

KLOET, 1994).

Há a hipótese que a ativação prolongada desse eixo pode deteriorar a

saúde e pode promover um elo entre o estresse mental e a doença física (doença

29

periodontal, por exemplo). A secreção de hormônios do estresse prejudica a

defesa do hospedeiro e ajuda no crescimento de organismos oportunistas no

sulco gengival (GOYAL et al., 2013). Doenças sistêmicas, tais como gastrite e

colite ulcerativa também estão ligadas ao estresse. Assim, o impacto do estresse

sobre a saúde periodontal não é apenas pela sua presença ou ausência, mas o

tipo, duração e como um indivíduo lida com ele. Os indivíduos sob estresse

tendem a adotar mudanças comportamentais, como da má higiene bucal,

tabagismo, apertamento ou ranger de dentes (GOYAL et al., 2011).

O estresse pode mediar efeitos na imunidade e/ou no comportamento das

defesas do organismo, contribuindo com a etiologia e perpetuação da doença

periodontal. O estresse pode mudar a resistência dos tecidos do hospedeiro por

mecanismos autonômicos e endócrinos, resultando primariamente em aumento

dos níveis de catecolaminas e corticoides (FOUREAUX et al., 2014).

O estresse psicológico resulta em secreção de adrenalina e noradrenalina

das células da medula adrenal. Através da interação com receptores

adrenérgicos, noradrenalina e adrenalina mediam efeitos cardiovasculares e

metabólicos. Em amostras de sangue coletadas imediatamente antes e depois de

uma situação de estresse emocional, a concentração circulante de linfócitos T-

helper, células T citotóxicas (CD8 +) e as células natural killer (células NK), é

aumentada, mas uma hora depois é reduzida para os valores iniciais. Além disso,

os níveis plasmáticos de imunoglobulinas IgM, IgG e componente C3 do sistema

complemento são elevados depois de uma situação de estresse agudo. Além

disso, a liberação de neuropeptidos, tais como a substância P (SP) que causa

dilatação e aumento da permeabilidade vascular, tanto diretamente como através

do estímulo à liberação e à produção de eucosanoides (prostaglandinas e

leucotrienos) pelos mastócitos, também modulam a atividade do sistema

imunológico e a liberação de citocinas (GOYAL et al., 2013).

30

Durante uma resposta ao estresse, o hipotálamo secreta o fator liberador

de corticotropina (CRF), o que estimula ainda mais o córtex adrenal e induz a

produção e liberação de hormônios glicocorticoides. Esses glicocorticoides

exercem os seus principais efeitos supressores, reduzindo o número e atividade

(quimiotaxia, secreção e degranulação) de células inflamatórias, incluindo os

linfócitos, monócitos, macrófagos, neutrófilos, eosinófilos e mastócitos e

também inibem a produção de mediadores pró-inflamatórios, citocinas IL-1, IL-

2, IL-3, IL-6, fator de necrose tumoral (TNF), interferon-γ, de granulócitos e

monócitos e da cascata da resposta imunitária através da inibição da

apresentação de antígeno de macrófagos, proliferação de linfócitos, e a

diferenciação de células efetoras de linfócitos, tais como linfócitos auxiliares,

linfócitos citotóxicos, as células NK e as células formadoras de anticorpos B

(GOYAL et al., 2013).

Os dois hormônios do eixo HPA, hormônio corticotrófico (CRF) e

hormônio adrenocorticotrófico (ACTH), além de aumentarem e manterem a

resposta inflamatória (MOLON et al., 2014), também modulam separadamente a

atividade do sistema imunológico, regulando a produção de substâncias

sinalizadoras a partir de células do sistema imune (citocinas), tais como IL-1

pelos monócitos e bloqueando a ativação dos macrófagos. Eles também

promovem a proliferação de células B, mas inibem a produção de anticorpo

(GOYAL et al., 2013).

A periodontite é uma doença multifatorial causada primariamente por

microrganismos, mas é significativamente dependente da resposta do hospedeiro

à invasão bacteriana (MUWAZI et al., 2014; TSAI et al., 2014). Indivíduos com

comportamento inadequado frente ao estresse têm maior risco de desenvolver a

doença periodontal severa. O estresse está associado à higiene oral deficiente,

aumento da secreção de glicocorticoides que podem deprimir a função

31

imunológica, aumento da resistência à insulina e aumenta o risco da periodontite

(ALJEHANI et al., 2014).

Mousavi Jazi et al. (2013) mostraram que o estresse pode aumentar os

níveis de citocinas pró-inflamatórias IL-1β, IL-6 e IL-10 e diminuir a produção

de IFN-γ que pode induzir a resposta Th1, sugerindo que existe uma interação

entre os sistemas endócrino e a resposta imune a um estresse fisiológico. Além

disso, os indivíduos com transtornos de humor também tiveram uma resposta

inflamatória exagerada ao estresse psicológico em comparação com indivíduos

saudáveis. Nesses estudos o valor médio de IL-1β no grupo periodontite

agressiva foi observado como sendo cerca de duas vezes maior do que no grupo

de pacientes com periodontite crônica e cerca de quatro vezes mais elevada do

que o grupo saudável.

Os níveis totais de IL-1β, IL-6 e IL-8 são, significativamente, elevados

em pacientes com doença periodontal, quando comparados com indivíduos

saudáveis. Adicionalmente, a gravidade do estresse tem uma forte relação com a

quantidade de IL-1β, tanto em pacientes com periodonte agressiva como crônica

(GIANNOPOULOU et al., 2003).

2.4 Probióticos e doença periodontal

A Food and Agriculture Organization (FAO) e World Health

Organization (WHO) definiram como probióticos os organismos vivos que

quando administrados em quantidades adequadas conferem benefícios à saúde

do hospedeiro (FOOD AND AGRICULTURE ORGANIZATION – FAO /

WORLD HEALTH ORGANIZATION - WHO, 2002).

O objetivo do tratamento periodontal é reduzir a infecção dos tecidos

periodontais através de rigorosa educação para higiene oral e pelo tratamento

mecânico (raspagem e alisamento corono-radicular e/ou cirurgia periodontal),

32

associados à terapia antimicrobiana química incluindo a administração sistêmica

de antibióticos nas formas crônicas graves e formas agressivas de periodontite

(HUCK et al., 2011).

Porém, o biofilme apresenta um problema terapêutico à medida que

oferece resistência à terapêutica microbiana convencional (MANCL; KIRSNER;

AJDIC, 2013). Teughels et al. (2011) investigaram o uso dos probióticos

influenciando a microbiota e a saúde periodontal e concluíram que os

probióticos podem melhorar a saúde periodontal pela interação microbiológica

ou pela interação imunomodulatória.

Assim, a identificação de probióticos úteis, o estabelecimento da dose

mais apropriada e o veículo para seu uso são áreas de investigação ativa. Embora

os probióticos estejam nos estágios iniciais da pesquisa científica e de sua

aplicação, eles aparentam ser uma ferramenta promissora (HAYTAC et al.,

2014). O impacto dos probióticos na saúde oral é relativamente novo com várias

pesquisas surgindo (KARUPPAIAH et al., 2013).

Os mecanismos de ação dos probióticos variam de acordo com a

linhagem ou combinações de linhagens específicas utilizadas, a presença de

prebióticos e da condição de tratamento, bem como a fase do processo da doença

em que o probiótico é administrado. Existem discussões comuns emergentes nos

estudos sobre os mecanismos de ação dos probióticos e numerosas hipóteses têm

sido propostas, incluindo:

a) a inibição da adesão de patógenos, colonização e a formação do

biofilme;

b) a indução de expressão de proteínas citoprotetoras na superfície das

células hospedeiras;

c) inibição de colagenases e redução de moléculas de inflamação

associadas;

33

d) a estimulação e modulação da resposta imune do hospedeiro, por

exemplo, por redução da produção de citocinas pró-inflamatórias

através de ações sobre NFkB aumentando a produção de citocinas

anti-inflamatórias tais como IL-10;

e) modulação da proliferação celular e da apoptose;

f) destruição ou inibição do crescimento de patógenos através da

produção de bacteriocinas ou outros produtos, tais como o ácido ou

peróxido, que são antagônicos em relação a bactérias patogênicas;

g) os probióticos também podem modificar o ambiente, através da

modulação do pH e/ou o potencial de oxidação-redução, que pode

comprometer a capacidade de agentes patogênicos de se tornarem

estabelecidos (GUPTA, 2011).

Nos estudos de Teughels et al. (2011) foram avaliados os efeitos de

pastilhas de Lactobacillus reuteri contendo probióticos como um adjunto para

raspagem e alisamento radicular (RAR). Após 12 semanas de tratamento, todos

os parâmetros clínicos foram significativamente diminuídos, com destaque para

a redução da profundidade da bolsa, ganho de inserção nas bolsas moderadas e

profunda e redução da contagem de Porphyromonas gingivalis.

Um estudo prévio do nosso grupo avaliou a influência do uso do

probiótico Bacillus subtilis (CH201) administrado oralmente e os níveis de perda

óssea alveolar foram significativamente reduzidos em animais com doença

periodontal que receberam probiótico (MESSORA et al., 2013).

No trabalho de Maekawa e Hajishengallis (2014), animais com doença

periodontal e tratados com L. brevis CD2 mostraram significativa diminuição da

perda óssea e menor expressão do fator de necrose tumoral e interleucina-1β, IL-

6 e IL-17A quando comparados com o tratamento controle.

34

2.5 Probióticos e imunidade

O trato gastrintestinal é um local onde a microbiota e antígenos são mais

expostos ao sistema imune (VITETTA et al., 2013). Tem sido sugerido que a

composição da microbiota intestinal esteja associada a condições alérgicas,

doenças inflamatórias intestinais, câncer, diabetes, doenças cardiovasculares e

dislipdemia (GOMES et al., 2014).

A microbiota do trato gastrintestinal é altamente estruturada e exerce

ampla influência protetora, estrutural, metabólica e imune dentro do intestino e

de forma sistêmica. A microbiota comunica-se com o sistema imune do

hospedeiro e, além disso, sinaliza vias de interação com órgãos tais como fígado,

músculos e cérebro, compreendendo uma série de ligações metabólicas

hospedeiro-microrganismos (VITETTA et al., 2013).

Os probióticos têm sido mostrados como moderadores em uma

variedade de funções fisiológicas do trato gastrintestinal que incluem controle

regulador sobre as respostas imunes, a função de barreira epitelial e proliferação

celular (GOMES et al., 2014; VITETTA et al., 2013).

A resposta do sistema imune, fatores genéticos e fatores ambientais

afetam o risco de desenvolvimento de doenças periodontais (MESA et al., 2014),

dependendo de como o hospedeiro responde à microbiota, tanto comensal como

patógena (EBERSOLE et al., 2013).

35

3 CONSIDERAÇÕES GERAIS

O uso de probióticos e suas aplicações vêm ganhando espaço. Existe um

aumento de evidências de que a espécies de probióticos podem ser benéficas à

saúde oral e sistêmica. Mais estudos serão necessários para otimizar o uso, dose,

administração e quantificar a extensão dos benefícios, bem como entender a

habilidade de sobrevivência da bactéria, o crescimento e o mecanismo de ação

sobre as diferentes doenças em que os organismos são usados. Foi observado

que o estresse crônico diminuiu os efeitos dos probióticos diminuindo os efeitos

imuno-modulatórios nos tecidos periodontais e ainda o uso de probiótico

protegeu o tecido intestinal.

36

REFERÊNCIAS

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46

SEGUNDA PARTE – ARTIGOS

ARTIGO 1

Effect of Probiotic Administration on the Immune Response: A Systematic

Review of Experimental Models in Rats

Silva, V. O.; Foureaux, R. C.; Araujo, T. S.; Peconick, P.; Zangeronimo, M.

G.; Pereira, L. J. Effect of Probiotic Administration on the Immune Response.

Brazilian Archives of Biology and Technology, Curitiba, v.55 n.5: pp. 685-694,

Sept/Oct 2012.

47

Effect of Probiotic Administration on the Immune Response: A Systematic

Review of Experimental Models in Rats

Viviam de Oliveira Silva1, Renata de Carvalho Foureaux1, Thiago

Santos Araujo1, Ana Paula Peconick2, Marcio Gilberto Zangeronimo1

and Luciano José Pereira1

1Departamento de Medicina Veterinária; Setor de Fisiologia e

Farmacologia; Universidade Federal de Lavras; Lavras – MG – Brasil.

2Departamento de Medicina Veterinária; Setor de Medicina Veterinária

Preventiva; Universidade Federal de Lavras; Campus Universitário, C.P.:

3037; 37200-000; Lavras – MG - Brasil

ABSTRACT

The probiotic influence on the immune system, especially under pathogenic challenge conditions, still remains controversial. To address this, a systematic review of current studies concerning the efficacy of probiotics on the immune response of rats subjected to experimental challenges was conducted. The survey was conducted using PubMed, ISI Web of Science and Scielo databases. Only studies which tested probiotics in vivo in rats were included. The experimental design, methodological quality, and results of the articles were analyzed. In total 21 articles were selected for this study. The most commonly used microorganisms in the experiments were those of the genus Lactobacillus, which was reported in 12 articles. The second most often used genus was Bifidobacterium (B. animalis and B.longum). In general, the probiotics use against experimental pathogenic challenges was successful: 86% of the selected articles reported a beneficial effect on the immune response associated with the use of probiotics.

Keywords: immunity, probiotics, rats, dietary supplements

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1 INTRODUCTION

It is well known that the nutrition, through a series of complex

interactions, is able to improve the health status of the animals. In animal

production, several substances have been used as growth promoters, including

probiotics, which are live microorganisms that improve the microbial balance in

the gastrointestinal tract, thereby increasing the efficiency with which the

nutrients are used. In other areas, probiotics have been used for preventive

purposes, to inhibit the proliferation of microorganisms that cause

gastrointestinal disturbances (Chaucheyras-Durand et al. 2008; Vanderpool et al.

2008; Mountzouris et al. 2009; Chaucheyras-Durand and Durant 2010;

Maragkoudakis et al. 2010).

By definition, probiotics are microorganisms that are regulated as

dietary supplements when ingested in sufficient quantities, have beneficial

effects on the health of the host (FAO 2002; Budiño et al. 2005; Siró et al.

2008; Tsubura et al., 2009). Most probiotics contain bacteria of the genus

Lactobacillus and Bifidobacterium (Brizuela et al. 2001; Peran et al. 2006;

Zeng et al. 2009; Bloise et al. 2010; de Roock et al. 2010).

However, certain bacteria of the genus Enterococcus (Maragkoudakis et

al. 2010), Leuconostoc and Streptococcus (Zanini et al. 2007) and yeasts, such

as Saccharomyces cerevisiae and Saccharomyces boulardii (Baptista et al.

2005; Generoso et al. 2010) can be considered to be probiotic microorganisms.

Numerous studies have demonstrated the effectiveness of these

microorganisms at improving the intestinal health of the animals and, thereby,

their metabolic and physiological status Brizuela et al. 2001). Besides the

direct effect of probiotics on the adherence of pathogenic bacteria in the

intestinal epithelium, several studies have also correlated probiotic

49

administration with the positive effects on the immune response in animals

(Borchers et al. 2009; Amit-Romach et al. 2010; Generoso et al. 2010; Fink

2010) and humans (Nomoto 2005; Salminen et al. 2005; Lomax and Calder

2009). Other benefits identified in in vitro studies include significant

inhibition of infection by L. monocytogenes (Corr et al., 2007); strong

induction of IL-12 and TNF-α in monocytes and cultured human peripheral

blood mononuclear cells (PBMC) (Fink 2010); and inhibition of the growth

of C. albicans (Verdenelli et al., 2009), among others. However, no consensus

exists in the literature on the preventive or therapeutic use of probiotics to

improve the immune system’s ability to defend against different infectious

agents.

Animal models, such as rats, are often used to simulate the physiological

and pathological mechanisms in vivo. Results are then extrapolated to other

species, which cannot be directly investigated, due to ethical, financial and/or

facilities management issues, or simply because of a lack of physical space

(Fagundes and Taha 2004; Da Matta 2010). Thus, detailed studies on a single

species are necessary for comparative analysis. Therefore, the objective of the

present study was to conduct a systematic review of the efficacy of probiotics

on the immune response in rats.

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2 MATERIALS AND METHODS

Research strategy

An electronic search of the PubMed database

(http://www.ncbi.nlm.nih.gov) was conducted in October 2010, using the

following keywords: immunity, probiotics, rats. To confirm the findings and

obtain supplementary studies, a similar strategy was employed for the ISI Web of

Science database (http://apps.isiknowledge.com) and Scielo database

(http://www.scielo.org/php/index.php), using the same keywords (also in

Portuguese and Spanish, when applicable).

Study Selection

For the present review, only in vivo studies using probiotics and rats

were selected. Studies conducted on mice, rabbits, guinea pigs, or other types of

animal models were excluded.

No restrictions were made for the type of probiotic used in the study,

administration form, or administration period against an experimental challenge

(for prevention and treatment). Additionally, no date, language or number of

animals were restricted as selection criteria.

Data extraction and Quality criteria

Two researchers conducted article searches separately, and

independently verified the compliance of the selected papers with the inclusion

criteria. In the cases of divergence between the papers, all the criteria were

reviewed and discussed. Table 1 displays the data related to the experimental

design of the retrieved articles. After study selection, quality analysis was

conducted and scores were assigned to specific scientific criteria as described in

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Table 2. Selection criteria were defined to evaluate both the protective effects

of probiotics in relation to the immune system and the methodological

quality of the selected articles.

However, not all the parameters used were scored on the quality scale

(such as animal strain, type of microorganism used and evaluated technique,

among others), but were taken into consideration as they were relevant to the

subsequent discussion. The scientific criteria used were adapted from other

systematic reviews (Noli and Auxilia 2005; Negre et al. 2009; Pereira et al.

2010). The parameters were classified as either adequate (score: 2) or

unclear/partially adequate (score: 1). The following parameters were scored:

• Sample number: Studies with sample groups containing ≥ 6

animals received a score of 2 and studies with less than 6 animals

per group received a score of 1.

• Randomization: Studies reporting nonrandomized experiments or

studies for which the degree of randomization was not clearly

described in the text received a score of 1, while studies using

randomized experimental designs received a score of 2.

• Control group: Studies that included a control group received a

score of 2, while studies that did not include a control group or did

not clearly mention a control group in the text received a score of 1.

• Blind evaluation: Studies which included blind assessments in their

experimental design received a score of 2, while studies whose

experimental designs did not include blind assessments, or for

which blind assessments were not clearly reported in the text

received a score of 1.

• Interference factors: Studies that did not evaluate interference

factors received a score of 1, while studies which considered

52

additional factors, such as stress, hormonal evaluation, and

variations between the males and females received a score of 2.

• Pathogenic challenge: Studies which did not include an

experimental challenge received a score of 1, while studies which

subjected the animals to an experimental challenge received a

score of 2.

The maximum total score was 12 points.

53

3 RESULTS

An initial search of the PubMed database retrieved 24 articles. Of these,

three were excluded because were conducted in humans or mice; one evaluated

the isolated action of prebiotics;three others were also excluded because they

were literature reviews. Thus, of the initial 24 articles retrieved, 18 were

selected for this study.

A search of the ISI Web of Science database also retrieved 24 articles,

eight of which were duplicates of articles retrieved from PubMed. Of the 16

remaining articles, five were excluded because they were studies on humans,

sows and piglets, prebiotics, or were performed in vitro; two others were

excluded because they did not evaluate probiotics, and six more were excluded

because they were literature reviews. Therefore, three additional papers were

selected from this search. A search of the database Scielo did not identify

additional articles. Thus, in total 21 articles met the inclusion and exclusion

criteria, and were selected for this review. Table 1 presents a summary of the

selected studies.

The following rat lineages were used in the studies included in this

review: Wistar, Lewis, Sprague Dawley and Fischer. The combinations of two

distinct lineages of rats were also used, as well as the combinations of rats with

Balb-C mice. In three studies, the authors did not report the rat lineages used.

Bacteria from the genus Lactobacillus were the most commonly used

microorganisms in the selected studies, and were reported in 57% of the papers.

The second most common genus was Bifidobacterium (B. animalis and B.

longum). The combinations of microorganisms were used in 38% of the

papers, such as Lactobacillus helveticus + Streptococcus thermophilus or

Streptococcus thermophilus + Lactobacillus acidophilus + Bifidobacterium

lactis, among others.

54

In 48% of the articles, probiotics were administered in the feeding,

while in 38% of the studies, probiotics were administered by gavage. Other

forms of administration, such as water or castor oil were also mentioned. There

was a large variability in the amount of colony forming units (CFU) used among

the surveys, and no consensus technique emerged, even among the studies

dealing with the same species of bacilli. The duration of probiotic administration

(e.g. before, during or after experimental challenge) also varied considerably:

probiotics were administered both before and after the challenge in 33% of the

studies; only during the challenge in 14%; and only after the challenge in 19%.

In 19% of the studies, probiotics were administered throughout the study period,

independent of the timing of the experimental challenge. Other studies

administered probiotics only a few days before the animals were killed, in the

pre- and post-operatory period.

Of the 21 selected articles, only one reported having conducted a blind

evaluation (So et al. 2008), while 16 articles reported randomization of the

sample. The number of animals per group ranged from 1 to 32, although three

papers did not report the number of animals used per experimental group.

A total of 90% of the articles induced a pathogenic challenge: 38%

introduced an intestinal challenge (e.g. colitis or tumors, among others); 9.5%

introduced encephalomyelitis; 9.5% induced liver injury; 4.8% induced

respiratory allergies; 4.8 % induced arthritis; 4.8% challenged the animals with

Escherichia coli; 4.8% induced ischemia and infusion; 4.8% induced

intracerebroventricular cannulation; and 4.8% were challenged by the

introduction of air pockets into the back of the animals. In 4.8% of the

papers, both encephalomyelitis and respiratory allergy were induced

simultaneously (Ezendam et al. 2008).

With respect to the interference factors 24% of the articles separated

male and female groups, while 5% used the models of stress. However, the

55

vast majority (71% of the articles) did not report any interference factor. In

the work of Laudanno et al. (2008), both the sexes (male/female) and stress

were evaluated. All the studies used control groups.

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4 DISCUSSION

Literature reviews are useful to the scientific community in general, and

can provide significant insight into a particular research field, since they enable

a more complete view of current results. In addition, they can suggest the best

protocols to be employed and/or future directions for research (Snodgrass

2006). The present literature review on the efficacy of probiotics at improving

the immune response in rats focused on targeting which therapeutic protocols

were associated with the best (or more promising) results in this species,

and could be used as a guide to future studies attempting to reproduce these

experiments in other species.

Research using animal models are important, especially given the

limitations of investigating certain diseases directly in humans, which often

involves the ethical issues and/or risks related to the disease under study.

Diseases which can be induced in animal models have the potential to reveal

the pathological mechanisms that can be extrapolated to humans, increasing the

understanding of human disease. Thus, the use of animal models can help

overcoming numerous research limitations and often provides causal

relationships more quickly. For these reasons, experimentation on animal

systems often represents the first step in many research projects (Taha and

Fagundes 2004; DaMatta 2010).

According to Nomoto (2005), excessive use of the antibiotics can

induce an imbalance in the intestinal microbiota, encouraging the emergence

of antibiotic-resistant bacterial infections, and, at the same time, reducing the

possible activation of the immune system prior to infection. Because of this

problem, interest in the use of probiotics as a complement to antibiotics has

been growing.

57

Although it is known that probiotics have different properties and

functions, the mechanisms by which individual probiotics act in a host are

not fully understood. As described in the literature, probiotics are assumed to

act via several mechanisms, including: a) competitive exclusion, where

probiotics compete with the pathogens for fixation sites and nutrients, thereby

temporarily preventing the pathogenic action; b) production of antimicrobial

substances, such as bacteriocins, hydrogen peroxide and volatile organic

acids; c) induction of direct changes in the immune response, through immune

stimulation of residing cells in the enteric tract, which then initiate activation

of macrophages, increasing phagocytosis; and d) modulation of enzyme

activity by changing the microbial metabolism (Audisio et al. 2000; de Vrese

et al. 2001; Ogawa et al. 2001; Cross 2002; Puupponen-Pimia et al. 2002;

Hamilton-Miller 2004; Boirivant and Strober 2007; Gillor et al. 2008;

Borchers et al. 2009; Ng et al. 2009; Rijkers et al. 2010; Yan and Polk, 2010).

Of the 21 articles selected, 86% reported the beneficial effects from the

administration of probiotics on the immune response in rats. Two studies, one

conducted by Baken et al. (2006) and one by Guitard et al. (2006) reported

unsatisfactory results from the use of probiotics, suggesting that further studies

were necessary. Baken et al. (2006) induced autoimmune encephalomyelitis,

the same challenge experiment used by Maassen et al. (2008), who concluded

that probiotics could suppress this disease. Ezendam et al. (2008) observed a

significant reduction in the duration of clinical symptoms, and an improvement

in weight gain versus the control group. Guitard et al. (2006) investigated the

effect of probiotic administration on the development and progression of an

experimental parasite infection (cryptosporidiosis) in lactating rats. Although

the rats administered probiotics tended to display faster parasite clearance

than the controls, no significant effect was observed in terms of weight gain,

parasite burden, mucosal damage or cytokine kinetics in the mucosa during the

58

course of the infection. Overall, these authors found that daily administration of

probiotic mixtures containing Lactobacillus casei was not able to eradicate the

parasite in their experimental model. However, differences in probiotic strains

and dosages could justify the discrepancies between these studies.

The animals underwent intestinal challenge in 48% of the assessed

studies, and all responded positively to the use of probiotics, with the exception

of Guitard et al. (2006). The immunostimulant effect associated with probiotic

administration could be related to the ability of these microorganisms to interact

with Payer’s patches and intestinal epithelial cells, thereby activating the

mucosal immunity by stimulating the plasma cells, IgA secretion and migration

of intestinal T cells (Park et al. 2002; de Vrese et al. 2005).

Of the articles investigating induced respiratory allergies (n=4 articles),

only two reported a positive response. However, probiotics were found to

increase the phagocytic activity of alveolar macrophages, suggesting that they

could act systemically by inducing the secretion of mediators which could then

stimulate the adaptive immune system (Cross 2002).

Significant differences were observed with respect to the doses of

probiotics used. However, no differences in results were noted between the

highest (Beaulieu et al. 2007) and lowest administered doses (Aguilar-

Nascimento et al. 2006): both showed positive immune responses. The

immune response to the use of probiotics was also not dependent on the time

of administration: positive responses were noted when probiotics were

administered before pathogen challenge, during the challenge, or both before

and after the challenge.

However, no trend was found among the studies analyzed regarding the

type of microorganism used for the treatment, preventing the establishment of a

general protocol. Lactobacillus were used against several different types of

pathogenic challenges, including encephalomyelitis (Baken et al. 2006;

59

Maassen and Claassen 2008); colitis (Amit-Romach et al. 2010); laparotomy

with colon anastomosis (Aguilar-Nascimento et al. 2006); and E. coli infection

(Ishida-Fujii et al. 2007), among others. With the exception of Baken et al.

(2006), all of these articles reported satisfactory results associated with the

probiotic administration. However, numerous other microorganisms were also

used in the analyzed studies, both alone and in combination. This variation

probably stemmed from the fact that the objective of the research was to

generally stimulate the immune response of the animals, not to evaluate the

specific infections. No relationship was identified between the type of probiotic,

pathogenic challenge and the effectiveness of probiotic administration.

Only one of the papers analyzed was conducted by blind assessment.

However, 71% of the articles included a randomized experimental design (the

remaining 29% did not clearly state if the study was randomized or not).

Use of blind assessments and randomized evaluations improved the reliability

of scientific works, by preventing study investigators from knowing which

treatment was administered and in the case of randomized trials, distribution

was done randomly (Snodgrass 2006; Taylor and Yildirim 2011).

60

5 CONCLUSION

In the studies assessed in this review, the administration of probiotics has

been shown to be associated with a positive induction of the immune response in

the presence of a wide range of experimental pathogenic challenges. Therefore,

further studies should be encouraged in this field in order to develop new

protocols with respect to the microorganism type, dosage and the timing of

probiotic administration for specific illnesses.

ACKNOWLEDGEMENTS

This work was supported in part by FAPEMIG, CNPq, CAPES and the

Postgraduate Program in Veterinary Sciences of the Federal University of

Lavras, Brazil.

61

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Table 1 - Summary of the Selected Studies. 1 2 3 4 5 6 7 8 9 10 11 12 13 A Wistar Lactobacillus

helveticus and Streptococcus thermophilus 106

CFU B Wistar Streptococcus

thermophilus, Lactobacillus acidophilus and Bifidobacterium lactis 109 CFU

Y Pre- and post- operative period

Y Before the

challenge and throughout the experiment

30 Y U Y U Laparotomy with colon anastomosis

6 Y U Y N Induction

of colitis

Intestine (colon) Intestine (colon and lower end of the ileum)

Evaluation of the IGA, total protein, albumin and globulin; analysis of DNA content by the method Gyles and Meyers. Evaluation of colonized tissues by real-time PCR. Morphology of the colon and damaged tissue were histologically evaluated.

C Lewis, Lactobacillus casei N Before the 16 Y U Y U EAE induction Ears Isolation and proliferation ofWistar 109 CFU challenge (epidermis) or lymph nodes, IL-4 and IFN-γ

and Balb/c

and throughout the experiment

central nervous system

by ELISA, cytokine through standard curves of recombinant IL-4 or IFN-γ, analysis of gene expression in liver and thymus tissue, as well as frozen MLN; analysis of the amount of RNA by spectrophotometry and RNA integrity by gel electrophoresis; microarray analysis

D Wistar and

Lewis

Lactobacillus kefiranofaciens 6 x 1011 CFU

N Throughou t the experiment

5 N U Y Y N - ELISA and blood cell count

E Sprague Dawley

Lactobacillus sp. 109 CFU

U Before the challenge and 9, 3 and 10 days after challenge

3 Y U Y N Cecum perforation for polymicrobial infection

Cecum Intestine histology; counting bacterial colonies; Backlight analysis; serum TNF analysis by ELISA.

F Wistar Lactobacillus casei U Before and 6 Y U Y N Listeria Gastro- Bacteriological analysis; liver2x109 CFU. after the monocytogenes intestinal tract and spleen histological

G Wistar Lactobacillus 2x109

CFU

challenge

N After the

challenge

(sensitization caused by oral infection)

4 Y U Y U Listeria

monocytogenes infection.

and visceral organs

Spleen and liver

analysis; ALT levels and concentration of total serum bile acids by a Beckman Synchron CX7, cell-mediated immunity measured using the DTH assay Liver and spleen bacteriological analysis and measurement of L. monocytogenes specific DTH.

H Sprague Dawley

Bifidobacterium longum 1×1010 CFU

N From birth until the end of the experiment

10 U U Y U N - RNA concentration by spectrophotometry, reverse transcription, RT-PCR, cytokine and immunoglobulin

(by ELISA) (Cont. …)

70

(Cont. Table 1) 1 2 3 4 5 6 7 8 9 10 11 12 13 I Lewis

and Balb/c

J Lewis and Balb/c

K Albino

Lactobacillus casei 2 - 4x108 CFU or 1 - 2x109 CFU

Bifidobacterium animalis 1x109 CFU

Lactobacillus sp.

N Before the challenge and throughout the experiment

N Before the

challenge and throughout the experiment

N Throughout

8 Y U Y Y Allergy Induction

16 Y U Y Y OVA

(respiratory allergy) or EAE

6 Y U Y Y Induction of

Lymphocytes in the lungs and ovalbumin- specific cytokines in the spleen Lung or central nervous system Gastro-

Specific ovalbumin IgE and IgG1 titres in sera were determined by ELISA. Th1 and Th2 cytokines were measured in the supernatants of spleen cells that were cultured with ovalbumin; IL-4, IL-5, IL-10, IL-13 and IFN-γ analysis. OVA-specific antibodies; Cytokine; IgE ova-specific (by ELISA) Protein levels were determined

rats 108 CFU the experiment

diarrhea (castor oil used as a laxative)

intestinal tract by the method of Buiret; blood cell count

L Sprague Dawley

Lactobacillus casei, Lactobacillus bulgaricus, Streptococus thermophilus, Lactobacillus acidophilus, Lactobacillus plantarum and Bifidobacterium infantis 2x107 CFU or 4x108 CFU

Y From the second day of the experiment until sacrifice

1 U U Y U Cryptosporidi osis

Small intestine (cecum)

Estimated amount of parasites in the mucosa of the cecum by Ziehl-Neelsen staining and C. parvum by real-time PCR, histological analysis of the cecum; IFN-γ, IL-10 and TNF- α

M U Lactobacillus casei 5x1010 CFU or 1x1011 CFU

N Before the challenge and throughout the experiment

10 Y U Y N Infection with E. coli

U IgA (ELISA), cytotoxicity of NK cells, macrophages, TNF- α, IL-6 and IL-12

O Fisher and Balb/c

Lactobacillus paracasei 5 x 108

CFU

N After the challenge

5 U U Y N Air bags (injection of sterile air)

Back of the animal

PMN accumulation and phagocytic activity of these cells, IFN-γ, TNF-α and IL-10 (ELISA), histopathology; immunohistochemistry

P Wistar Lactobacillus Y For 3 days 10 Y U Y Y Indomethacin Gastro- Percentage of damaged areabrevis, Lactobacillus plantarum, Streptococcus faecalism and Bifidobacte-rium brevis 6 x 108 CFU

before sacrifice

intestinal tract (macroscopically); Histology of the gastric mucosa, ileum and colon, immunohistochemistry of lymphocytes B (CD 20) and T (CD 4 +)

Q U Lactobacillus plantarum and Lactobacillus murines CFU (UC)

N During the challenge

U U U Y N EAE Central nervous system

Cytokines and DNA

R Sprague Dawley

Lactobacillus acidophilus, L.. helveticus and Bifidobacterium CFU (UC)

Y Throughout the experiment

U Y U Y U Azinomethane (colon carcinoma)

Colon Analysis of the proliferation rate of the mucosa, mesenteric lymph nodes were removed from rats for analysis of intestinal immune system markers, ACF determination; tumor detection

S Wistar Lactobacillus paracasei 3 x 107

CFU

N During the challenge

7 Y U Y U Ischemia and reperfusion

Liver Hepatic microcirculation, liver histology, Western blotting analysis, plasma assessment; bacteriological evaluation in

the small intestine(Cont. …)

71

(Cont. Table 1) 1 2 3 4 5 6 7 8 9 10 11 12 13 T Fischer Lactobacillus

rhamnosus and Bifidobacterium lactis 5x108 CFU or 5.5x108 CFU

Y U 32 Y U Y U Azinomethane (colon carcinoma)

Colon Immunofluorescence of lymphocyte subpopulations through the spleen and MLN, flow cytometry analysis; IL-10 and IFN-γ by ELISA

V Lewis Lactobacillus casei 2x1010 CFU

N After induction and during the whole experiment

U Y Y Y Y Induction of rheumatoid arthritis (collagen type II)

Ankle (foot) Histopathological analysis of the hind paws; cytokines by RT-PCR, IgG (ELISA), TNF- α, IL-10 and Foxp3 by FACS Calibur Flow Cytometer

X Sprague Dawley

Lactobacillus acidophilus 2.5 x 108 CFU

N After the challenge

7 U U Y N ICV cannulations

Brain tissue Histopathology; immunohistochemistry; mRNA and cDNA (RT-PCR), positive colonies were confirmed by DNA sequencing, Western- blotting of the intestines and retroperitoneal adipose tissue.

A: Aguilar-Nascimento et al. 2006; B: Amit-Romach et al. 2010; C: Baken et al. 2006; D: Beaulieu et al. 2007; E: Bu et al. 2006; F: de Waard et al. 2002 a; G: de Waard et al. 2002 b; H: Dong et al. 2010; I: Ezendam and van Loveren 2008; J: Ezendam et al. 2008; K: Flore et al. 2010; L: Guitard et al. 2006; M: Ishida-Fujii et al. 2007; O: Kourelis 2010; P: Laudanno et al. 2008; Q: Maassen and Claassen 2008; R: Marotta et al. 2003; S: Nardone et al. 2010; T: Roller et al. 2004; V: So et al. 2008; X: Sousa et al. 2008; CFU: colony forming unit; U: Unclear; Y: YES; N: NO; IgA: immunoglobulin A; IgE: immunoglobulin E; IgG1: immunoglobulin G; DNA: deoxyribonucleic acid; RNA: ribonucleic acid; mRNA: Messenger ribonucleic acid; ELISA: Enzyme Linked Immunosorbent Assay; PCR: Polymerase Chain Reaction; RT-PCR: reverse transcription polymerase chain reaction; EAE: experimental autoimmune encephalomyelitis; IL-4: interleukin-4; IL-5: interleukin-5; IL-6: interleukin-6; IL-10: interleukin-10; IL-12: interleukin-12; IL-13: interleukin-13; ICV: intracerebroventricular; MLN: mesenteric lymph nodes; PMN: polymorphonuclear leukocyte; ACF: aberrant crypt foci ALT: alanine aminotransferase; DTH: delayed-type hypersensitivity ; Th1: T helper cell type 1; Th2: T helper cell type 2; IFN-γ: interferon-gamma; TNF-α: tumor necrosis factor-α; OVA: ovalbumin; NK: natural killer; 1: author and year of publication; 2: lineage; 3: microorganisms used; 4: association of microorganisms; 5: period of probiotic administration; 6: number of animals per experimental group°; 7: randomization; 8: blind assessments; 9: control group: 10: interference factors°°, 11: pathogenic challenge; 12: tissue where the challenge was induced; 13: technical evaluated. °Studies in which the "n" experimental varied, was considered the smallest n; °°Stress, hormone assessment, gender, etc.

72

Table 2 - Evaluation cr iteria and scores for the selected articles.

Author Mean number of Type of Control Blind Interference Pathogenic Total animals per gr oup* assay** group*** assessmentd+ fators++ challenge+++

Ezendam and van 2 2 2 1 2 2 11 Loveren 2008 Ezendam et al. 2008 2 2 2 1 2 2 11 Laudanno et al. 2008 2 2 2 1 2 2 11 So et al. 2008 1 2 2 2 2 2 11 Aguilar-Nascimento 2 2 2 1 1 2 10 et al. 2006 Amit-Romach 2 2 2 1 1 2 10 et al. 2010 Baken et al. 2006 2 2 2 1 1 2 10 Flore et al. 2010 2 2 2 1 1 2 10 Bu et al. 2006 2 2 2 1 1 2 10 Ishida-Fujii et al. 2007 2 2 2 1 1 2 10 Roll er et al. 2004 2 2 2 1 1 2 10 de Waard et al. 2002a 2 2 2 1 1 2 10 Beaulieu et al. 2007 2 1 2 1 2 1 9 Marotta et al. 2003 1 2 2 1 1 2 9 Nardone et al. 2010 2 1 2 1 1 2 9 Sousa et al. 2008 2 1 2 1 1 2 9 de Waard et al. 2002b 1 2 2 1 1 2 9 Kourelis 2010 1 1 2 1 1 2 8 Guitard et al. 2006 1 1 2 1 1 2 8 Maassen and Claassen 1 1 2 1 1 2 8 2008 Dong et al. 2010 2 1 2 1 1 1 8

*Scores for the sample number were 1 (less than 6 animals/group) and 2 (6 or more animals/group) ** Nonrandomized experiments or when randomization was not described clearly in the text (score 1) and randomized experiments (score 2) *** Studies without control groups or those which did not clearly mention a control group in the text (score 1) and studies with a control group (score 2) + Experiments without blind assessments or those in which blind assessments were not clearly reported in the text (score 1) and experiments with blind assessments (score 2) ++ Studies that did not evaluate interference factors (score 1) and studies which evaluated additional factors such as: stress, hormonal evaluation, variations between males and females (score 2) +++ Studies in which animals were not subjected to experimental challenge (score 1), and studies in which animals were subjected to an experimental challenge (score 2).

73

ARTIGO 2

Effects of probiotic therapy on metabolic and inflammatory parameters of

rats with ligature-induced periodontitis associated with restraint stress

Foureaux, R. C.; Messora, M. R.; Oliveira, L. F. F.; Napimoga, M. H.; Pereira,

A. N. J.;, Ferreira, M. S.; Pereira, L. J. Effects of Probiotic Therapy on

Metabolic and Inflammatory Parameters of Rats With Ligature-Induced

Periodontitis Associated With Restraint Stress. Journal of Periodontology,

Chicago, v. 85, n. 7, p. 975-983, July 2014.

74

Effects of probiotic therapy on metabolic and inflammatory parameters of

rats with ligature-induced periodontitis associated with restraint stress

Running title: Probiotics, periodontitis and stress

Renata de C. Foureaux, DDS, MS*

Michel R. Messora, DDS, PhD†

Luiz Fernando F. de Oliveira, DDS†

Marcelo H. Napimoga, DDS, PhD‡

Andressa N. J. Pereira*

Matheus S. Ferreira*

Luciano J. Pereira, DDS, PhD*

Corresponding author:

Prof. Dr. Luciano José Pereira:

e-mail address: [email protected]

Department of Physiology and Pharmacology - Federal University of Lavras –

UFLA, Lavras / MG – Brazil

Source of Support: National Council for Research and Technological

Development (CNPq - Process 475468/2010-4, Brasília, DF, Brazil) and the

Research Foundation of the State of Minas Gerais (FAPEMIG, Belo Horizonte,

MG, Brazil). There is no relationship between any author and a commercial firm

that may pose a conflict of interest.

Word count: 3976

Number of figures: 3

Number of tables: 2

Summary Sentence: Stress seems to affect the action of probiotic agents,

modulating their effects in intestinal and periodontal tissues.

75

Author affiliations: * Physiology and Pharmacology Department, Federal University of Lavras -

UFLA, Lavras / MG, Brazil. † Department of Oral and Maxillofacial Surgery and Periodontology, School of

Dentistry of Ribeirao Preto, University of Sao Paulo - USP, Ribeirão Preto, SP,

Brazil. ‡ Laboratory of Immunology and Molecular Biology, Faculty São Leopoldo

Mandic, Campinas, SP, Brazil.

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Abstract

Background: This study evaluated the effects of Probiotic Therapy (PT) in rats with ligature-induced periodontitis associated with restraint stress. Methods: Sixty-four rats were divided into: control (C), stress (STR), probiotic (PROB), periodontal disease (PD), STR-PROB, STR-PD, STR-PROB-PD and PROB-PD goups. The probiotic was added to the drinking water for 44 days. PD was induced by a ligature. In STR groups, the animals were subjected to restraint stress for 2.5 hours during 30 days. Results: PD increased alveolar bone loss (p<0.05). PD also increased levels of Cyclooxygenase-2 (COX-2), serum C-terminal telopeptide (CTX), p38 mitogen-activated protein kinase (p38), receptor activator of NF- κ B ligand (RANKL) and decreased levels of osteoprotegerin (OPG). Stressed rats presented high levels of C-peptide, corticosterone and glucose (p<0.05). In general, the presence of stress reduced the expression of CTX and p38 (p<0.05). PT reduced alveolar bone loss in unstressed animals. It also decreased expression of CTX and induced increased expression of OPG in unstressed animals with PD. However, PT was not effective in preventing bone loss or altering the expression of inflammatory markers in stressed animals. PT decreased the number of inflammatory cells in the periodontal tissue (p<0.05). Groups with stress and PD presented decreased villous height and crypt depth. Stress seemed to prevent part of probiotic beneficial effects on small intestine. Conclusion: Based on the methodology used, PT may reduce tissue destruction resulting from PD in unstressed rats. The protocol used for restraint stress decreased the immuno-modulatory effects in intestinal and periodontal tissues of probiotics. Keywords: Inflammation, periodontal diseases, physical restraints, animal models.

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1 Introduction

Periodontal disease (PD) is characterized by a chronic inflammatory

process of tooth supporting tissues.1-4 The most common form, chronic

periodontitis, affects 35% to 60% of the adult population in developed

countries,5 and severe forms are estimated to affect 5 to 15% of the world’s

population.6 It was reported that one in two Americans aged 30 or older has

periodontits.7 Besides being the most common cause of tooth loss, there is some

evidence that periodontal infection can also be involved in systemic disease

processes, such as cardiac abnormalities and preterm low birth weight.8-10

The presence of biofilm is essential for initiating PD. However, the

clinical course of the condition depends on host-pathogen interactions.6 Bacterial

products start a local response in the gingival tissue 11 leading to resorption of

alveolar support bone, mobility and consequent tooth loss.1 Nevertheless, the

presence of biofilm alone accounts for only a small proportion (20%) of the

variations in expression of PD. 12 The main contributor to the destruction of hard

and soft tissues is the result of activation of an immune-inflammatory host

response to bacterial aggression.13 Acquired risk factors and environmental

factors (e.g. diabetes mellitus, smoking and stress) as well as genetically

transmitted characteristics (e.g. polymorphisms for interleukin-1) may

exacerbate the inflammatory response.13

Thus, in addition to microorganisms, stress can mediate immune and/or

behavioural effects on the body's defences, contributing to the aetiology and

perpetuation of chronic periodontitis.14 Stress can change the resistance of the

host tissue by autonomic as well as endocrine mechanisms, resulting primarily

in increased levels of catecholamines and corticosteroids, reduced

microcirculation in the gums and salivary flow, interference with the normal

functioning of neutrophils and lymphocytes and facilitation of bacterial invasion

and tissue damage.15,16

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In this context, probiotics (live microorganisms that confer health

benefits17 – e.g. bacteria of the genus Lactobacillus, Enterococcus, Bacillus and

Bifidobacterium)18 appear as new adjuvants for controlling PD.19

As far as we are concerned, there is no previous study evaluating the

effect of Probiotic Therapy (PT) in association to PD and stress. In a previous

study from our group, it was shown that PT reduced epithelial attachment and

alveolar bone loss in rats with ligature-induced periodontitis.20 Since probiotics

can interfere both with microorganism load and with the immune response, their

systemic application is of interest.

The impact of stress on exacerbating inflammatory processes has a

particular role in the management of PD.4,14,21 Due to the diversity and

complexity of the interactions of probiotic species with the immune system, a

careful selection process should be conducted before using them in clinical

trials.22 Since similar immune-inflammatory mechanisms occur in periodontal

tissue and intestinal mucosa, it is believed that the action of probiotics in the oral

cavity is analogous to that described in intestinal mucosa. Bacillus subtilis along

with other probiotics is known to be valuable for prevention of enteric

infections. We hypothesized that its use in drinking water should adhere to and

colonize part of the oral and intestinal bacteria with beneficial effects on

intestinal and periodontal tissues even under stress situations. Therefore, this

study aimed to evaluate the effects of Probiotic Therapy (PT) in rats with

ligature-induced periodontitis associated with restraint stress

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2 Materials And Methods

All procedures were approved by the Ethics Committee on Animal

Experimentation (CEUA – UFLA protocol number 036/10). A total of 64

healthy adult male rats (Rattus norvegicus Albinus, Wistar) weighing

approximately 200 g were used in this study. A completely randomized design

in a 2x2x2 factorial scheme (stressed or not, with periodontal disease or not,

treated or not with probiotics) was employed. After acclimatizing for 7 days, the

animals were divided into eight groups with eight animals each: control, stress

(STR), probiotic (PROB), periodontal disease (PD), STR-PROB, STR-PD,

PROB-PD and STR-PROB-PD. The animals were housed in individual

metabolic cages. The room was kept at a temperature of 22 ± 2°C with light-

dark cycles of 12/12 h. Food and water were supplied ad libitum throughout the

experimental period.

The rats were immobilized in plastic containers adapted to their

size/weight. 21 The restriction of movements was maintained for 2.5 hours per

day over 30 days (Figure 1).18 The immobilization was performed in a quiet

room and always at the same time to avoid additional stress or interference with

the circadian cycle.

Probiotic product based on Bacillus subtilis (CH201)§ was orally

administered for 44 days (1.5 x 108 colony-forming units CFU/mL daily).23 PD

was induced by a ligature protocol in both mandibular first molars. The animals

received general anaesthesia with intraperitoneal injection of 10 mg/kg xylazineơ

and 80 mg/kg¶ ketamine. Once anesthetized, the animals received a ligature with

cotton# according to the methodology described by Holzausen et al.24

All animals were killed after 44 days and their tissues used for

subsequent analyses. The rats were decapitated with a guillotine. Blood samples

were collected for assessment of glucose levels (by colorimetric assay),

80

corticosterone** and C-peptide†† (by enzyme immunoassay - ELISA) using

commercial kits.

After collecting blood, the jaws were removed and fixed in buffered

formalin in a volume of liquid at least 30 times greater than the removed tissue.

The right hemi-mandibles were destined for histological slide preparation for

counting inflammatory cells, while the left side of each mandible was used for

morphometric analysis and also to collect tissues for semi-quantitative assays of

proteins by Western Blotting. These analyses are described below.

Inflammatory cell counts

The right hemi-mandibles were demineralized in 10% EDTA solution

for 60 days,25 paraffin embedded, then cut into a half series of slices with

thickness 6.0 µm in the mesio-distal direction. The sections were fixed on slides,

dewaxed and stained with hematoxylin and eosin. Their images were captured

under a light microscope. Before the analysis, criteria were established in order

to conduct a more objective evaluation of inflammatory infiltrates in the

interproximal sub-epithelial connective tissue between the distal root of the first

mandibular molar and the mesial root of the second mandibular molar. The

counts of inflammatory cells such as eosinophils, lymphocytes, plasma cells and

macrophages26 in the interproximal papilla between the first and second molars

were performed using a computer program ‡‡.

The analyses were performed by two calibrated examiners who were

blinded to the experimental groups. A second sample was measured again 48

hours after the first. A paired t test was used to calculate the intra-examiner

error. A Pearson correlation analysis between the data obtained by the two

examiners was also performed. P values > 0.05 in the paired t test and r> 0.90 in

the Pearson correlation test were considered in estimating the feasibility of the

proposed method.

Western blotting

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Quantification of proteins: p38 mitogen-activated protein kinase (p38),

Cyclooxygenase-2 (COX-2), receptor activator of NF- κ B ligand (RANKL) and

osteoprotegerin (OPG) were visualised from total protein extracts of gingival

tissues. Samples of 40 µg of total protein were added in a buffer containing

glycerol and SDS DTT, heated and denatured at 95°C for 5 min, and loaded onto

SDS polyacrylamide gels. Proteins on the gel were separated by electrophoresis

in 10 mM Tris-HCl buffer subjected to a voltage of 100 V for 90 min.

Subsequently they were electro-transferred to 0.2 mM nitrocellulose membranes

(300 mA for 60 min). The membranes were then incubated with antibodies

against a housekeeping protein (α-tubulin) used as a positive control. Images

obtained on radiograph films were scanned by a chemiluminescent

documentation system and further analysis was performed by densitometry of

the bands using specific software.

ELISA for serum C-terminal telopeptide (CTX) protein

CTX levels were determined in capture ELISA assays using microtiter

plates§§ coated for 24 h at 4oC with 2 µg/ml of goat IgG anti-rat CTX in

carbonate-bicarbonate buffer, pH 9.6. Unless stated elsewhere, all antibodies

were affinity purified and obtained from Rheabiotech (Campinas/SP, Brazil).

ELISA reactions were performed in 100 µl volumes. After coating, the plates

were washed 3 times with a solution of 0.9% NaCl, 0.05% Tween 20 and 0.02%

NaN3, then blocked for 1 hour at room temperature with phosphate buffer

(PBS), pH 7.5, and 0.01% BSA. After a new series of washes, gingival proteins

were applied in triplicate and the plates were incubated for 2 hours at room

temperature. After additional washes, the plates were incubated for 2 hours at

room temperature with a 1:5000 dilution of biotin-conjugated goat IgG anti-rat

CTX. The plates were then washed and incubated for 1 h with a 1:500 solution

of streptavidine. The plates were washed again and the reactions were revealed

82

by incubation with the TMB substrate. Colour development was measured at

450 nm in an ELISA plate readerơơ.

Morphometric analysis of periodontal tissue

The left hemi-mandibles were fixed in 10% formalin solution.

Subsequently, they were immersed in 30% hydrogen peroxide for 2 hours and

the soft tissues were removed with gauze, followed by staining with 1%

methylene blue for 30 minutes in order to differentiate bone from teeth and

enhance the visibility of the cement-enamel junction (CEJ). Using a

stereomicroscope## with a colour video camera mounted on it and coupled to a

computer, the lingual surfaces of the defleshed jaws were recorded in a

standardized manner (20 x magnification). The distance between the alveolar

bone crest (ABC) and the CEJ (ABC–CEJ, mm) was measured on the three

distal root surface of the first molar, using image analysis software ##.

Histomorphometric analysis of small intestine

Tissues samples from the small intestine (duodenum, jejunum and

ileum) were collected and fixed in neutral formalin for 48 hours. The samples

were routinely processed and embedded in paraffin. Serial sections, 6 µm thick,

were obtained. The sections were stained with H&E for analysis by light

microscopy. Villous height was estimated by measuring the vertical distance

from the villous tip to the villous-crypt junction level for 10 villi per section;

crypt depth was estimated by measuring the vertical distance from the villous-

crypt junction to the lower limit of the crypt for 10 corresponding crypts per

section, as described previously. 20

83

Statistical analysis

Analysis of variance (Three-way ANOVA) was performed to compare

the means between groups in a 2x2x2 factorial design (with and without

probiotic, with and without stress, with and without periodontal disease). When

F values indicated significant differences in the interactions between these

factors, they were deployed. The number of inflammatory cells were analysed

by Kruskal-wallis test among all groups. Analyses were performed using the

SigmaStat 3.1 program (Sigma StatStatistical Package Software Inc., USA) and

Statistical Analysis System (Statistical Analysis System Institute - SAS Institute

(1996). The significance level was set at p <0.05.

84

3 Results

Animals subjected to chronic restraint stress showed higher levels of

plasma corticosterone, C-peptide (referencing insulin production) and glucose (P

<0.05) compared to unstressed animals, indicating that the model was adequate

for inducing stress. The use of probiotic and induction of PD did not interfere

with these parameters (p> 0.05) (Figure 2).

The animals subjected to stress showed decreased levels of CTX, and

placement of the ligature induced an increase in this marker. However, there

were significant interactions between stress and PD, leading to higher levels of

CTX. The use of probiotic decreased the levels of CTX in unstressed animals

with PD (p<0.05). In stressed animals with PD, PT had no effect (Figure 3).

The expression of COX-2 was higher in the unstressed groups with PD.

The stress protocol did not alter the expression of this enzyme in relation to the

respective controls (p <0.05), nor did PT. Similarly, in the groups with PD there

was increased expression of p38 (p <0.05), but this expression was reduced in

the presence of stress (p<0.05) (Figure 3). In addition, there was a significant

interaction effect between probiotic and PD (p<0.05), where the use of probiotic

in animals with ligature leaded to smaller amounts of p38.

In groups with PD there was increased expression of RANKL (p <0.05).

The use of probiotic did not affect the expression of this protein, nor the stress

induction. Nonetheless, significant interaction between Probiotic and PD was

found (p<0.05), where the use of probiotic mitigated the effects of PD. OPG

expression was decreased in the groups with PD (p <0.05). There was no

significant influence of stress. PT induced an increase in the expression of this

protein in unstressed animals with PD (p <0.05) (Figure 3).

The number of inflammatory cells was higher in the groups with PD and

stress (p <0.05). The use of probiotic significantly decreased the numbers of

85

these cells (p<0.05), as shown in Figure 3. Groups PD, STR-PD, PD-PROB and

STR-PROB-PD presented foreign bodies on the surface of the gingival

epithelium, loss of the interdental papillae, apical migration of the junctional

epithelium, interdigitation towards the underlying connective tissue and

presence of moderate to severe mononuclear inflammatory infiltrate in

subepithelial connective tissue.

There was no difference in bone loss in stressed and unstressed animals

that received ligature (p>0.05) (Table 1). PD increased alveolar bone loss (p<

0.05) and probiotic had a positive effect against this loss in unstressed animals

(p<0.05). However, it was observed that in stressed groups there was no

preventive effect of probiotic on bone loss.

It was observed that the animals with stress and PD presented decreased

villous height and crypt depth. Significant statistical interactions were found

between PD and Probiotics for duodenum and jejunum tissues (in general

probiotics promoted greater villi height and crypt depth), and also between stress

and PD in jejunum (stress associated to PD promoted shorter villi

height)(p<0,05). (Table 2).

86

4 Discussion

Previously, beneficial effects of using a probiotic to prevent bone loss

caused by PD in unstressed rats were observed in a study from our group.20

Those results suggested that orally administered probiotics may produce benefits

both by passage through the oral cavity and by modulating its oral mucosal

immunity systemically.27

There is evidence that colonization of the gut by probiotics can cause

beneficial systemic effects, providing protection against disease at distant sites.28

Thus, it is suggested that beneficial effects of the probiotic may include

preventing adhesion of pathogens to host tissues while passing through the oral

cavity. Additionally, stimulation and modulation of the immune system,

reducing the production of pro-inflammatory and increasing the production of

anti-inflammatory cytokines,29 enhancing the integrity of the intestinal barrier

and increasing the production of mucins, as well as eliminating or inhibiting

pathogen growth by producing bacteriocins or other products such as acids and

peroxides30 and decreasing luminal pH.31

However, it was not known if these effects were still present under

stressful conditions. In this study, the beneficial effects of probiotics were

minimized in animals subjected to restraint stress. The relationship between

stress and PD progression is still controversial in the literature.4,32,33 The feasible

mechanism involved in the pathogenesis of stress in PD is related to increased

production of glucocorticoids (corticosterone) and catecholamines. Animals

subjected to stress showed corticosterone levels significantly above their

respective controls without stress, as observed in previous studies, indicating the

validity of the proposed model.4 Stress alone did not induce any significant

change in periodontal tissues, as demonstrated in previous studies.,32,33 However,

animals subjected to restraint stress did not show more pronounced bone loss

87

than unstressed animals in groups with induced PD, despite the numbers of

inflammatory cells being significantly higher in those groups. These differences

can be justified by variations in restraint periods, manner of restraint 34 and

gender susceptibility, since females are more prone to stress models than

males.35 It was observed that bone loss in animals undergoing ligature and the

stress protocol showed slightly less bone loss than the unstressed groups

(although not significantly less), indicating a possible anti-inflammatory effect

of plasma corticosterone levels in stressed animals, reducing bone loss in

animals with respect to induction of the disease alone.4

Although stress is considered an important risk factor for PD,36 studies

evaluating the influence of probiotics in stressed rats have not yet been

performed. It is speculated that stress may produce important effects in the

gastrointestinal tract, which may have affected the modulating role of probiotics

on the immune system in the performed model. Increased secretion of

norepinephrine and corticosterone may influence the interactions of host cells

involved in the protection of intestinal mucosa with commensal bacteria that

inhabit the mucosal surface37 by changing the effect of probiotics in the presence

of stress.

Studies in rodents have shown that exposure to stressors can lead to

diseases of the small and large intestine, including altered ion secretion and

increased epithelial permeability. Prolonged exposure to stress can also induce

low-grade inflammation, cause ultrastructural epithelial abnormalities and alter

interactions between intestinal bacteria, allowing greater microbial

translocation.38 Additionally, stressful events promote changes in the

temperature and acidity of the gastrointestinal tract,39 possibly having a negative

impact on the viability of the probiotic. Thus, this set of physiological reactions

to stress in the gastrointestinal tract may have contributed to the inefficiency of

88

the bacilli in stressed animals, as also evidenced by the present intestinal

findings.

In this study, the blood glucose levels of animals submitted to the stress

were increased. This result was due to activation of the hypothalamic-pituitary-

adrenal axis and the sympathetic nervous system, which results in increased

production of catecholamines and glucocorticoids, respectively, with a

consequent increase in glucose production by the liver and decreased use by

tissues.40 With respect to insulin levels, in the present study we chose to quantify

them indirectly, through the measurement of plasma C-peptide. Since the

principal stimulus for insulin secretion is hyperglycemia,41 the increases in C-

peptide in stressed animals were expected.

With regard to inflammation at periodontal sites, COX-2 expression was

higher in the groups with PD. The stress protocol did not alter the expression of

this enzyme in relation to these groups’ respective controls, corroborating the

findings of Rettori et al.4 for prostaglandin E2 (PGE2), the main product of COX-

2. The production of PGE2 from the activity of COX-2 is elevated in subjects

with PD compared to healthy subjects, corroborating our findings.42 The use of

probiotics did not influence the expression of this protein, nor did stress.43

The p38 MAPK kinase is a subfamily that plays roles in adaptation,

homeostasis and the stress response and may also be involved in inflammatory

responses. Besides JNK, p38 is responsible for inflammation and the

development of stress-induced signalling. The main function of the p38α MAPK

in RANKL-induced osteoclastogenesis was determined to be the formation of

precursor cells of macrophages/osteoclasts.44 In the present study it was

observed that after PD induction in unstressed groups, increased expression of

p38 protein was seen, corroborating Aquino et al.45 where activation of p38 was

significant on the fifth day after induction by PD ligation as well as injection of

LPS. It was also observed that the groups subjected to stress induction showed

89

reduced expression of p38, suggesting that stress may have modulated the

expression of this protein.

The proteins RANKL and OPG modulate osteoclastogenesis. RANKL is

a key molecule in osteoclast activation and OPG is a decoy receptor for

RANKL. Therefore, the relative proportion of OPG on RANKL determines the

speed and intensity of bone resorption mediated osteoclastogenesis.46 In the

present study, groups with PD had higher amounts of RANKL, corroborating the

study of Jabbar et al.47 The findings with the OPG also agree with the literature

showing a decreased expression of this protein in groups with PD.48 The use of

probiotic induced increased expression of this protein in non-stressed animals

with PD.

The measurement of CTX has been considered as a marker of bone

resorption. In this study, ligature placement induced an increase in this marker

and animals subjected to stress showed decreased levels of CTX, corroborating

previous findings. 47,49 The use of probiotic also decreased levels of CTX in

unstressed animals with PD. It has been suggested that reducing CTX can

determine the degree of suppression of osteoclasts.50 Thus, it is suggested that

the use of probiotics may favour lower levels of CTX, decreasing osteoclast

activity and thus preventing periodontal bone loss.

In general, the effects of the probiotic were mostly modified under stress

conditions employed in this experiment. However, the number of inflammatory

cells was still influenced by the use of probiotics even under restraint stress.

Further studies are essential to exploit these mechanisms of action of probiotics

in periodontal tissues and other stress protocols should also be investigated.

90

5 Conclusions

Based on the methodology used, PT may reduce tissue destruction resulting

from PD in unstressed rats. Stress seems to affect the action of probiotic agents,

modulating their effects in intestinal and periodontal tissues.

Footnotes: § Bioplus 2B (CH Hansen (Hørsholm, Denmark)

ơ Rompum, Bayer Animal Health, São Paulo, SP, Brazil

¶ Dopalem, Agribands, Paulínia, SP, Brazil. # Coats-Corrente, São Paulo, SP, Brazil. ** DetectX ® Corticosterone Enzyme Immunoassay– Arbor Assays, New

Orleans, LA, USA ††RayBio ® C-Peptide Immunoassay Protocol, RayBiotech, Inc., Norcross

(Georgia), USA ‡‡ Cell B - Olympus CX31, Olympus Optical Co., Tokyo, Japan §§Costar 3590, Fisher Scientific, PA, USA

ơơMicroplate Reader / Model 3550, Bio Rad, Promega Corporation, Fitchburg,

Wisconsin, USA. ¶¶ Leica MZ6, Leica Microsystems GmbH, Wetzlar, Germany ## Imagelab - DiraconBio Informática Ltda., Vargem Grande do Sul, SP, Brazil

Acknowledgements:

The National Council for Research and Technological Development (CNPq -

Process 475468/2010-4, Brasília, DF, Brazil) and the Research Foundation of

the State of Minas Gerais (FAPEMIG, Belo Horizonte, MG, Brazil). CH Hansen

(Hørsholm, Denmark).

91

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Figure 1 Time schedule of experimental period

98

* Differs significantly from the respective control (without stress) by F test (P <0.05)

Figure 2 Means and standard deviations of plasmatic corticosterone, C-peptide and glucose levels

99

Different capital letters A,B indicate difference between groups with and without periodontal disease by F test (P<0.05) Different small letters a,b indicate difference between groups with and without probiotic therapy by F test (P<0.05) *Differs significantly from the respective control (without stress) by F test (P<0.05) Figure 3 Means and standard deviations of CTX, Cox-2, p-38, RANK-L, OPG

expression and number of inflammatory cells evaluated on periodontal tissues

100

Table 1 Mean values of morphometric analysis rat mandible Probiotic

Stress Periodontal

disease Without With

Morphometric analysis - 0.72 (0.04) a 0.74 (0.01)

Without + 1.02 (0.02) bB 0.86 (0.04)A

- 0.80 (0.05)a 0.78 (0.13)a With

+ 1.00 (0.01)b 0.91 (0.11)b

- : without periodontal disease; + : with periodontal disease Different capital letters A,B indicate difference between groups with and without periodontal disease by F test (P<0.05) Different small letters a,b indicate difference between groups with and without probiotic therapy by F test (P<0.05)

101

Table 2 Means and standard deviations of the villous height and crypt depth in small intestine sections, with comparisons among groups

Villous height Crypt depth

Stress PD Probiotic Probiotic

without with without with

Duodenum

without - 1176 (72,20) A 1306 (66,37) A 559 (5,85) ABb 657 (32,22) Ba

+ 1118 (44,91) Ba 1068 (31,89) Ab 634 (38,87) Ab 775 (66,40) Aa

with - 1095 (77,72) B 1143 (93,76)BC 610 (46,59) A 629 (23,67) B

+ 984 (81,89)C 1031 (50,85)C 536 (40,51) Bb 614 (49,43) Ba

Jejunum

without - 759 (45,64) A 882 (59,90) A 478 (26,43) A 424 (61,34) AB

+ 528 (51,65) B 672 (83,27) BC 269 (48,41) Bb 463 (100,94) Aa

with - 503 (38,03) B 647 (113,49) BC 302 (41,95) B 365 (79,70) B

+ 614 (51,97) B 522 (45,96) C 279 (53,84) B 352 (24,63) BC

Ileum

without - 546 (26,61) A 532 (55,50) A 350 (36,19) A 353 (19,37)

+ 552 (44,98) A 544 (31,62) A 340 (26,28) A 360 (48,88)

with - 449 (38,05) B 464 (34,04) B 311 (30,48) Bb 351 (18,47) a

+ 488 (39,56) AB 529 (28,77) A 289 (10,96) ABb 369 (19,94) a

PD: Periodontal Disease; - : without periodontal disease; + : with periodontal disease Different capital letters A,B indicate difference between groups with and without periodontal disease by F test (P<0.05) Different small letters a,b indicate difference between groups with and without probiotic therapy by F test (P<0.05)

102

ANEXOS

Certificado fornecido pela Comissão de Ética no Uso de Animais da

Universidade Federal de Lavras para a realização da pesquisa

103

Tabela 1A - Variável analisada: CTX Opção de transformação: Raiz quadrada - SQRT ( Y )

FV GL SQ QM F P>F

Probiótico (P) 1 0,0025 0,0025 0,0015 0,9697 Estresse (E) 1 0,8421 0,8421 0,5039 0,4852 Doença (D) 1 2,7504 2,7504 1,6458 0,2129 P*E 1 0,9580 0,9580 0,5733 0,4570 P*D 1 2,7434 2,7434 1,6417 0,2134 E*D 1 8,6288 8,6288 5,1636 0,0332 P*D*D 1 5,7813 5,7813 3,4596 0,0763 erro 22 36,7642 1,6711

CV % 29,79

Tabela 2A - Variável analisada: RANK_L Opção de transformação: Raiz quadrada - SQRT (Y)

FV GL SQ QM F P>F

Probiótico (P) 1 0,1689 0,1689 7,8303 0,0105

Estresse (E) 1 0,0463 0,0463 2,1452 0,1572

Doença (D) 1 0,0746 0,0746 3,4569 0,0764

P*E 1 0,0044 0,0044 0,2053 0,6549

P*D 1 0,1114 0,1114 5,1641 0,0332

E*D 1 0,0354 0,0354 1,6430 0,2133 P*D*D 1 0,0935 0,0935 4,3344 0,0492 erro 22 0,4746 0,0216

CV % 16,38

104

Tabela 3A - Variável analisada: OPG Opção de transformação: Raiz quadrada - SQRT ( Y )

FV GL SQ QM F P>F

Probiótico (P) 1 0,0194 0,0194 0,8155 0,3763

Estresse (E) 1 0,4662 0,4662 19,5995 0,0002

Doença (D) 1 0,0014 0,0014 0,0580 0,8119

P*E 1 0,0199 0,0199 0,8354 0,3706

P*D 1 0,0032 0,0032 0,1347 0,7171

E*D 1 0,0216 0,0216 0,9094 0,3506 P*D*D 1 0,0935 0,0935 3,9306 0,0600 erro 22 0,5233 0,0238

CV % 17,59

Tabela 4A - Variável analisada: COX_2 Opção de transformação: Raiz quadrada - SQRT (Y)

FV GL SQ QM F P>F

Probiótico (P) 1 0,0010 0,0010 0,0316 0,8606

Estresse (E) 1 0,4733 0,4733 15,0521 0,0008

Doença (D) 1 0,0090 0,0090 0,2855 0,5985

P*E 1 0,2113 0,2113 6,7208 0,0166

P*D 1 0,1246 0,1246 3,9628 0,0591

E*D 1 0,0060 0,0060 0,1908 0,6665

P*D*D 1 0,0935 0,0935 2,9732 0,0987

erro 22 0,6918 0,0314

CV % 16,43

105

Tabela 5A - Variável analisada: p38 Opção de transformação: Raiz quadrada - SQRT ( Y )

FV GL SQ QM F P>F

Probiótico (P) 1 0,0414 0,0414 5,3804 0,0300

Estresse (E) 1 0,2959 0,2959 38,4897 0,0000

Doença (D) 1 0,0075 0,0075 0,9714 0,3350

P*E 1 0,0584 0,0584 7,5945 0,0115

P*D 1 0,0482 0,0482 6,2725 0,0202

E*D 1 0,0294 0,0294 3,8289 0,0632

P*D*D 1 0,0935 0,0935 12,1607 0,0021

erro 22 0,1692 0,0077

CV % 10,99

Tabela 6A - Variável analisada: MORFOMÉTRICO Opção de transformação: Variável sem transformação ( Y )

FV GL SQ QM F P>F

Probiótico (P) 1 0,0049 0,0049 0,7716 0,3902

Estresse (E) 1 0,0065 0,0065 1,0222 0,3241

Doença (D) 1 0,2927 0,2927 46,0869 0,0000

P*E 1 0,0049 0,0049 0,7653 0,3921

P*D 1 0,0221 0,0221 3,4862 0,0766

E*D 1 0,0073 0,0073 1,1417 0,2980

P*D*D 1 0,0334 0,0334 5,2510 0,0329

erro 20 0,1270 0,0064

CV % 9,20

106

Tabela 7A - Variável analisada: GLICEMIA Opção de transformação: Raiz quadrada - SQRT ( Y )

FV GL SQ QM F P>F

Probiótico (P) 1 1,1365 1,1365 1,3344 0,2549

Estresse (E) 1 6,6290 6,6290 7,7833 0,0080

Doença (D) 1 0,0088 0,0088 0,0103 0,9197

P*E 1 0,1766 0,1766 0,2073 0,6514

P*D 1 1,3948 1,3948 1,6376 0,2080

E*D 1 2,6645 2,6645 3,1285 0,0846

P*D*D 1 0,0612 0,0612 0,0719 0,7900

erro 40 34,0677 0,8517

CV % 9,02

Tabela 8A - Variável analisada: CORTICOSTERONA Opção de transformação: Variável sem transformação ( Y )

FV GL SQ QM Fc Pr>Fc Estresse (E) 1 2523052,9000 2523052,9000 11,313 0,0020 Doença (D) 1 849139,6000 849139,6000 3,807 0,0598 Probiótico (P) 1 90820,9000 90820,9000 0,407 0,5279 E*D 1 136422,4000 136422,4000 0,612 0,4399 E*P 1 344844,9000 344844,9000 1,546 0,2227 D*P 1 751856,4000 751856,4000 3,371 0,0757 E*D*P 1 906010,0000 906010,0000 4,062 0,0523 erro 32 7136990,8000 223030,9625 CV% 58,45

107

Tabela 9A - Variável analisada: PEPTÍDEOC Opção de transformação: Variável sem transformação ( Y )

FV GL SQ QM Fc Pr>Fc

Estresse (E) 1 58140,6250 58140,6250 6,960 0,0128

Doença (D) 1 88642,2250 88642,2250 10,612 0,0027

Probiótico (P) 1 256800,6250 256800,6250 30,743 0,0000

E*D 1 186459,0250 186459,0250 22,322 0,0000

E*P 1 185096,0250 185096,0250 22,159 0,0000

D*P 1 316662,0250 316662,0250 37,910 0,0000

E*D*P 1 12075,6250 12075,6250 1,446 0,2380

erro 32 267296,8000 8353,0250

CV% 7,42

Tabela 10A - Variável analisada: DUODENO VILOSIDADES Opção de transformação: Variável sem transformação ( Y )

FV GL SQ QM Fc Pr>Fc

Estresse (E) 1 103369,9224 103369,9224 18,238 0,0002

Doença (D) 1 142857,4752 142857,4752 25,205 0,0000

Probiótico (P) 1 2184,1884 2184,1884 0,385 0,5391

E*D 1 641,2806 641,2806 0,113 0,7388

E*P 1 10543,0090 10543,0090 1,860 0,1821

D*P 1 38980,0435 38980,0435 6,877 0,0133

E*D*P 1 38243,0928 38243,0928 6,747 0,0141

erro 32 181372,8274 5667,9008

CV% 6,75

108

Tabela 11A - Variável analisada: DUODENO CRIPTAS Opção de transformação: Variável sem transformação ( Y )

FV GL SQ QM Fc Pr>Fc

Estresse (E) 1 25398,5760 25398,5760 13,352 0,0009

Doença (D) 1 6124,6350 6124,6350 3,220 0,0822

Probiótico (P) 1 57968,4276 57968,4276 30,473 0,0000

E*D 1 47630,7022 47630,7022 25,039 0,0000

E*P 1 7428,8953 7428,8953 3,905 0,0568

D*P 1 5785,4680 5785,4680 3,041 0,0908

E*D*P 1 263,3742 263,3742 0,138 0,7123

erro 32 60872,5904 1902,2684

CV% 7,00

Tabela 12A - Variável analisada: JEJUNO VILOSIDADES Opção de transformação: Variável sem transformação ( Y )

FV GL SQ QM Fc Pr>Fc

Estresse (E) 1 212738,2688 212738,2688 32,180 0,0000

Doença (D) 1 71123,0789 71123,0789 10,759 0,0025

Probiótico (P) 1 24802,8900 24802,8900 3,752 0,0616

E*D 1 59818,6230 59818,6230 9,049 0,0051

E*P 1 5902,2273 5902,2273 0,893 0,3518

D*P 1 37267,9725 37267,9725 5,637 0,0237

E*D*P 1 32796,8109 32796,8109 4,961 0,0331

erro 32 211547,7508 6610,8672

CV% 12,61

109

Tabela 13A - Variável analisada: JEJUNO CRIPTAS Opção de transformação: Variável sem transformação ( Y )

FV GL SQ QM Fc Pr>Fc

Estresse (E) 1 50944,6200 50944,6200 14,806 0,0005

Doença (D) 1 20485,1286 20485,1286 5,954 00204

Probiótico (P) 1 40168,3426 40168,3426 11,674 0,0017

E*D 1 7608,4947 7608,4947 2,211 0,1468

E*P 1 248,2530 248,2530 0,072 0,7900

D*P 1 59015,4286 59015,4286 17,152 0,0002

E*D*P 1 52146,4515 52146,4515 15,156 0,0005

erro 32 110103,6454 3440,7389

CV% 16,26

Tabela 14A - Variável analisada: ÍLEO VILOSIDADES Opção de transformação: Variável sem transformação ( Y )

FV GL SQ QM Fc Pr>Fc

Estresse (E) 1 36673,9248 36673,9248 24,840 0,0000

Doença (D) 1 9293,9619 9293,9619 6,295 0,0174

Probiótico (P) 1 744,5964 744,5964 0,504 0,4827

E*D 1 4672,5145 4672,5145 3,165 0,0847

E*P 1 3732,2376 3732,2376 2,528 0,1217

D*P 1 641,9214 641,9214 0,435 0,5144

E*D*P 1 225,2451 225,2451 0,153 0,6987

erro 32 47244,8250 1476,4007

CV% 7,48

110

Tabela 15A - Variável analisada: ÍLEO CRIPTAS Opção de transformação: Variável sem transformação (Y)

FV GL SQ QM Fc Pr>Fc

Estresse (E) 1 4370,8174 4370,8174 5,335 0,0275

Doença (D) 1 889,7205 889,7205 1,086 0,3052

Probiótico (P) 1 13279,8292 13279,8292 16,210 0,0003

E*D 1 1192,7916 1192,7916 1,456 0,2364

E*P 1 5829,5688 5829,5688 7,116 0,0119

D*P 1 116,3833 116,3833 0,142 0,7087

E*D*P 1 259,6412 259,6412 0,317 0,5774

erro 32 26214,9528 819,2172

CV% 8,40